The indictment accuses the Colombo family of orchestrating a two-decade scheme to extort an unnamed labor union that represented construction workers, using threats of violence to secure payments and arrange contracts that would benefit the crime family.

The charges are an ambitious effort by the U.S. attorney’s office in Brooklyn and the Federal Bureau of Investigation to take down one of the city’s five Mafia families. In addition to the union extortion scheme, which is the heart of the racketeering charge, the indictment charges several misdeeds often associated with the mob, including drug trafficking, money laundering, loan-sharking and falsifying federal labor safety paperwork.

Detention hearings for the defendants in Brooklyn federal court continued into the evening Tuesday, as they entered not-guilty pleas to the charges; prosecutors had asked the court to keep 10 of the defendants in custody.

“Everything we allege in this investigation proves history does indeed repeat itself,” Michael J. Driscoll, F.B.I. assistant director-in-charge, said in a statement. “The underbelly of the crime families in New York City is alive and well.”

Around 2001, prosecutors said, Vincent Ricciardo — a reported captain in the family, also known as “Vinny Unions” — began to demand a portion of a senior labor union official’s salary. When Mr. Ricciardo was convicted and imprisoned on federal racketeering charges in the mid-2000s, prosecutors said, his cousin continued to collect those payments.

Starting in late 2019, prosecutors said, the senior leadership of the Colombo family became directly involved in the shakedown, which extended to broader efforts to siphon money from the union: for example, manipulating the selection of union health fund vendors to contract with entities connected to the family, and diverting more than $10,000 each month from the fund to the family.

Andrew Russo, 87, who prosecutors describe as the family boss, is accused of taking part in those efforts, as well as a money-laundering scheme to send the proceeds of the union extortion through intermediaries to Colombo associates. He was among nine defendants charged with racketeering.

Mr. Russo appeared in court virtually from the hospital Tuesday; he is set to be detained upon his release, pending a future bail hearing.

The family’s infamous longtime boss, Carmine J. Persico, died in federal custody in North Carolina in March 2019.

Federal law enforcement learned of the extortion scheme about a year ago, prosecutors wrote in a court filing Tuesday; investigators gathered thousands of hours of wiretapped calls and conversations recorded by a confidential witness, wrote the prosecutors, who also described law-enforcement surveillance of meetings among the accused conspirators.

The authorities said they repeatedly captured Mr. Ricciardo and his associates threatening to kill the union official. “I’ll put him in the ground right in front of his wife and kids,” Mr. Ricciardo was recorded saying in June.

On another occasion cited by prosecutors in the memo seeking his detention, Mr. Ricciardo directed the union official to hire a consultant selected by the Colombo family, saying: “It’s my union and that’s it.” Prosecutors said his activities were overseen by a Colombo soldier and the consigliere who remains at large.

Much of the activity outlined in the indictment took place while the defendants were either in prison or on supervised release for prior federal mob-related convictions. Theodore Persico Jr., described as a family captain and soldier, was released from federal prison in 2020 and, despite a directive not to associate with members of organized crime, “directed much of the labor racketeering scheme,” prosecutors said.

Mr. Persico, 58, is set to inherit the role of boss after Mr. Russo, prosecutors wrote.

Several of the defendants were named in what prosecutors described as a fraudulent safety training scheme, in which they falsified state and federal paperwork that is required for construction workers to show they have completed safety training courses.

One of the defendants, John Ragano — whom prosecutors say is a soldier in the Bonanno crime family — is accused of setting up phony occupational safety training schools in New York, which prosecutors said were “mills” that provided fraudulent safety training certificates to hundreds of people.

In October 2020, prosecutors said, an undercover law enforcement officer visited one of the schools in Ozone Park, Queens, and received, from Mr. Ricciardo’s cousin, a blank test form and an answer sheet; weeks later, the agent returned to pick up his federal safety card and paid $500.

The purported schools were also used for meetings with members of La Cosa Nostra — the group of crime families commonly known as the Mafia — and to store illegal drugs and fireworks, according to the indictment.

Mr. Ragano wasn’t charged on the racketeering count, although prosecutors also sought his detention pending trial. In addition to the racketeering count, several defendants, including Mr. Ricciardo and his cousin, were charged with extortion, conspiracy, fraud and conspiracy to make false statements.

William K. Rashbaum contributed reporting.