President Trump instructed a broad swath of government agencies on Thursday to end collective bargaining with federal unions, a major escalation in his effort to assert more control over the federal work force.

Mr. Trump framed the order as critical to protect national security. But it targets agencies across the government, including the Departments of Defense, Veterans Affairs, State, Treasury and Energy, most of the Justice Department, and parts of the Departments of Commerce, Homeland Security, and Health and Human Services.

The American Federation of Government Employees, the largest federal workers union, estimated that the order would strip labor protections from hundreds of thousands of civil servants, and said it was preparing legal action.

“This administration’s bullying tactics represent a clear threat not just to federal employees and their unions, but to every American who values democracy and the freedoms of speech and association,” Everett Kelley, the union’s president, said in a statement. “Trump’s threat to unions and working people across America is clear: fall in line or else.”

Unions have been a major obstacle in Mr. Trump’s effort to slash the size of the federal work force and reshape the government to put it more directly under his control. They have repeatedly sued over his blizzard of executive actions, winning at least temporary reprieves for some fired federal workers and blocking efforts to dismantle portions of the government.

To claim authority to cancel the union contracts under the Civil Service Reform Act of 1978, Mr. Trump expanded the list of agencies exempt from provisions of laws governing federal labor relations for national security reasons. In doing so, he adopted an expansive view of national security, one that encompasses agencies like the Environmental Protection Agency, the National Science Foundation, the U.S. International Trade Commission and the Federal Communications Commission.

The American Federation of Government Employees said Mr. Trump’s order was illegal.

After Mr. Trump signed the order, the affected agencies filed a lawsuit on Thursday in Texas against the unions representing federal employees, seeking to rescind their collective bargaining agreements.

The government argued that the agreements “significantly constrain” the executive branch and hinder the president’s “efforts to protect the United States from foreign and domestic threats.” In the filing, the government contended that the Biden administration extended the labor agreements for five years shortly after Mr. Trump won the election in November. The government also raised concerns about the contracts’ return-to-work policies.

The executive order is the latest step in Mr. Trump’s wide-ranging effort to drastically overhaul the federal bureaucracy, which he has assigned Elon Musk and his Department of Government Efficient to oversee. Mr. Musk said in an interview that aired Thursday on Fox News that he was trying to reduce the federal deficit by $1 trillion, and to cut $4 billion every day.

Federal workers are already bracing for major new cuts. Mr. Trump signed an executive order aimed at dismantling the Education Department last week, and on Thursday, Robert F. Kennedy Jr., the secretary of health and human services, announced that he was laying off 10,000 employees as part of a broad reorganization.