Soon after President Trump returned to office last year, a moment inside the Justice Department sent a chill through parts of the federal bureaucracy.

Hector F. Ruiz, a seasoned civil rights attorney at the department, informed his team that he had received instructions to immediately open investigations into more than a dozen universities. According to people familiar with the meeting, the directive departed sharply from standard practice, which typically requires extensive fact-gathering before launching formal probes. Several investigators viewed the order as politically driven rather than evidence-based.

Within a matter of months, Mr. Ruiz and 17 lawyers from his unit resigned.

Despite the departures, the Trump administration pressed ahead. Federal agencies launched investigations into at least 75 colleges and universities, examining issues ranging from antisemitism to alleged student aid fraud. While the administration portrayed the campaign as necessary oversight, the schools involved have consistently denied wrongdoing, including those that eventually reached settlements. In several cases, federal judges ruled that the administration’s tactics violated the law.

The university investigations have become an early and vivid illustration of a broader strategy that has defined much of Mr. Trump’s second term: using the machinery of government to confront institutions he views as political adversaries. Administration officials argue that the effort has already produced results and should continue, particularly in reshaping what they describe as deeply “woke” academic cultures hostile to conservative viewpoints.

One of the administration’s most powerful tools has been the coupling of investigations with threats to federal funding. By signaling that grants and contracts could be frozen or revoked, the White House forced university leaders into a difficult choice between defending academic autonomy and absorbing potentially devastating financial losses.

The pressure has reshaped campus life in subtle but significant ways. Professors report avoiding certain topics that allies of the president have publicly criticized. Researchers worry that a single phrase in a grant proposal or lecture could trigger scrutiny from Washington. Institutions that once prided themselves on open inquiry now operate in an atmosphere of caution, with administrators, faculty and students increasingly on edge.

Six elite universities have opted to settle civil rights investigations rather than continue fighting. Collectively, they agreed to adopt parts of the administration’s agenda and to pay hundreds of millions of dollars either to the federal government or to projects supported by the White House. Harvard University has so far refused to settle and, as a result, has been subjected to 13 investigations across 10 federal agencies in the past year alone.

“There’s no question that institutions have been bent — processes, norms, even democratic infrastructure connected to higher education,” said Todd Wolfson, president of the American Association of University Professors. “They haven’t been completely broken yet, and that distinction matters.”

Encouraged by what they see as progress, Trump officials are preparing a second attempt to persuade colleges to sign a voluntary agreement outlining the administration’s principles. The first version of the proposal gained little traction. At the same time, the White House is exploring ways to enforce individual provisions even without university consent, according to May Mailman, a senior adviser for special projects.

Ms. Mailman said the administration’s pressure campaign exceeded even internal expectations, though she emphasized that universities were not being asked to transform into partisan platforms. Still, she acknowledged that the effort had driven widespread institutional change.

In interviews, Mr. Trump has repeatedly accused colleges of engaging in what he calls “reverse discrimination,” pointing to diversity initiatives aimed at increasing minority representation. While much of the daily enforcement has been delegated to senior aides, universities remain a personal fixation for the president.

Public opinion has given the administration some political cover. Surveys show growing dissatisfaction with higher education, fueled by concerns over student debt, job readiness and campus culture. Jewish advocacy organizations have raised alarms about antisemitism at some schools, while conservatives argue that dissenting views are routinely marginalized.

Some university leaders concede that reforms are necessary. Yet many say the administration’s confrontational tactics have alienated potential partners and resemble methods historically used by authoritarian governments to suppress independent institutions.

“The goal is to silence opposition,” said Corey Brettschneider, a political scientist at Brown University. “Universities, because they protect free thought, have become an obvious target.”

Inside the federal government, the aggressive strategy has unsettled even veteran officials. After Mr. Ruiz’s meeting, Justice Department leadership assembled a special team to investigate the University of California system. Lawyers were given just one month to demonstrate that antisemitism was widespread across the system’s 10 campuses and to pursue workplace discrimination claims.

Former Justice Department attorneys say the focus was narrowly political. Investigations centered on institutions in Democratic-leaning states, while complaints elsewhere stalled. Antisemitism allegations, they argued, gained traction only when tied to protests over Israel and Gaza.

The inquiry ultimately concluded that UCLA had tolerated antisemitism. To resolve the case, administration officials proposed a $1 billion fine — a figure Governor Gavin Newsom of California condemned as political extortion. University leaders warned such a penalty would jeopardize the institution’s mission and financial stability.

Harvard soon became another focal point. In April, the university sued the administration, alleging constitutional and procedural violations. The lawsuit angered Mr. Trump, who pushed aides to halt funding and drafted a letter threatening to bar the school from future grants — an effort later blocked by the courts.

Negotiations between Harvard and the White House have continued intermittently, though no agreement has been reached. Mr. Trump has publicly suggested that a deal is close, even as he continues to express personal resentment toward the university’s legal team.

Beyond formal investigations, many universities have taken preemptive steps to avoid confrontation. Several schools closed or rebranded diversity, equity and inclusion offices after the administration labeled them discriminatory. Others paused graduate admissions or tightened classroom rules on discussions of race and gender. In some cases, student publications and academic leaders were sidelined amid fears of retaliation.

At the University of Virginia, concerns about federal scrutiny contributed to the resignation of President James E. Ryan. After his departure, Justice Department investigations into the school were dropped.

Faculty members across the country say the impact has reached into classrooms and laboratories. At UCLA, reproductive biology professor Amander Clark said she now carefully edits grant proposals and public statements, worried that politically sensitive language could jeopardize funding for her entire institution.

“It’s incredibly stressful,” she said, noting that even minor wording choices now feel risky.

While the administration highlights settlement agreements as major victories, the deals themselves reveal limits to its leverage. None of the universities admitted wrongdoing, and many commitments are broadly defined. Some schools retained diversity offices even after pledging to end “unlawful” practices.

Federal judges have delivered the administration its sharpest setbacks. Courts in Boston and San Francisco ruled that funding freezes and investigative tactics amounted to unlawful coercion and ideological targeting. The administration has appealed both decisions.

Those rulings emboldened universities to resist a proposed “compact” that would have tied research funding to policies such as strict gender definitions, tuition freezes and caps on international enrollment. No institution ultimately signed on.

Still, the White House has continued pursuing its goals through other channels, including quiet changes to visa processing that favor schools with lower proportions of international students.

For many in higher education, the failed compact underscored the need to defend academic independence.

“There must be clear red lines,” said Christopher Eisgruber, president of Princeton University. “Universities have to retain control over teaching and research, regardless of political pressure.”

Even within the administration, tensions have emerged between those seeking headline-grabbing financial settlements and officials pushing for deeper ideological changes. Negotiations with Harvard reportedly stalled amid internal disagreements over how hard to press.

As global crises and domestic controversies compete for the president’s attention, some university leaders argue that reform is unavoidable, with or without White House pressure.

“We’ve drifted from our core mission,” said Daniel Diermeier, chancellor of Vanderbilt University. “If we don’t correct course, public trust will continue to erode.”

Where the confrontation between Washington and academia ultimately leads remains uncertain. But as Princeton’s president noted, the long-standing partnership between the federal government and universities has played a critical role in American prosperity.

“We should be finding a way forward,” he said, “not tearing that relationship apart.”