Vincent Asaro, 78, reputedly a senior member of the notorious Bonanno crime family, was among five suspects detained by FBI agents in separate early morning raids in New York.
"These 'goodfellas' thought they had a license to steal, a license to kill, and a license to do whatever they wanted," said FBI Assistant Director in Charge George Venizelos.
During a nine-minute appearance in US federal court, Asaro pleaded not guilty to extortion, murder and violence charges that US prosecutors say spanned a 45-year career in crime.
He was also charged with the 1969 murder of Paul Katz, a presumed informant who was strangled with a dog chain and whose body parts were discovered in a New York basement in June.
Asaro, who had triple bypass surgery in March, faces life in prison if convicted.
He stood slightly stooped, his hair swept back and casually dressed in a sweater as he entered his not-guilty plea before the packed courtroom in Brooklyn.
US Magistrate Judge Marilyn Go ordered him detained until further notice. Asaro's lawyer vowed to apply for bail.
In the biggest heist on US soil, armed mobsters stole $5 million in cash and nearly $1 million in jewels from a Lufthansa Airlines vault at New York's JFK on December 11, 1978.
The value of the booty today is estimated at around $20 million.
The theft became legendary after its alleged mastermind James Burke -- also known as Jimmy the Gent -- killed off members of the crew to avoid being shopped to the police.
US officials say Asaro, Burke and their co-conspirators each expected to receive approximately $750,000 in cash and large quantities of gold jewelry after the robbery.
Scorsese immortalized the criminal feat in his Oscar-winning 1990 movie "Goodfellas," long considered one of the best crime films of all time.
Burke, who died of cancer in prison in 1996, was the inspiration for Robert De Niro's character Jimmy Conway in the film.
Asaro appeared alongside his burly son Jerome, 55, who sported a blue hoodie and shaved head, John Ragano, 52, and Jack Bonventre, 45.
The other three men face up to 20 years behind bars if convicted. They have not been named in connection with the 1978 JFK heist.
A fifth defendant, 70-year-old Thomas Di Fiore, who was previously the highest-ranking member of the Bonanno family to be out of jail, is due to appear in court Friday.
The charges against the older Asaro date from January 1968 to June 2013.
He is accused of strangling Katz with Burke because they suspected he was cooperating with investigators. They buried his body in the basement of a vacant home.
In the mid-1980s, Asaro allegedly ordered Jerome and another individual to dig up Katz's body and move it to avoid detection by investigators.
Almost 35 years later, the FBI searched the Queens residence still owned by the Burke family and recovered some of Katz's remains in the basement.
A detailed 63-page detention memo lists the charges as racketeering, acts of murder, robbery, extortion, arson, illegal gambling, loansharking and assault.
Prosecutors claim to have witness testimony, physical evidence, DNA, photographic evidence spanning several decades and documentary evidence in the case.
They accuse the two Asaros of participating in additional armed robberies, including around $1 million in gold salts.
"Far from a code of honor, theirs was a code of violence and brute force. Those suspected of cooperating with law enforcement paid with their lives," said US Attorney Loretta Lynch.
"Neither age nor time dimmed Asaro's ruthless ways, as he continued to order violence to carry out mob business in recent months."