Bufalino was born on October 29, 1903, in Montedoro, Sicily.

Bufalino immigrated with his family to the United States through the Port of New York in 1906.

Bufalino settled in Buffalo, New York, where he became a criminal during his teenage years.

In the early 1920s, Bufalino started working with Joseph Barbara, another upstate New York bootlegger in Endicott, New York.

Bufalino married Carolina Sciandra, who came from a Sicilian Mafia family.

Bufalino worked alongside many Buffalo mobsters, some of whom would become top leaders in the Buffalo crime family and other future Cosa Nostra families along the East Coast of the United States. These relationships proved very helpful to Bufalino in his criminal career. Family and clan ties were important to Sicilian-American criminals; they created a strong, secretive support system that outsiders or law enforcement could not infiltrate.

Bufalino later moved to Kingston, Pennsylvania in 1940.

In the early 1950s, the Immigration and Naturalization Service tried to have Bufalino deported several times, but had never been successful over 15 years as the Italian government would not readmit him to the country.

Bufalino met truck driver Frank Sheeran in 1955, when Bufalino offered to help him fix his truck; Sheeran later worked jobs driving him around and making deliveries. Bufalino had introduced Sheeran to Teamsters International President Jimmy Hoffa. Hoffa, who became a close friend to Sheeran, used him for muscle, including the assassination of recalcitrant union members and members of rival unions threatening the Teamsters' turf.

A local state trooper named Edgar D. Croswell had been aware that Carmine Galante had been stopped by state troopers following a visit to Barbara's estate. A check of Galante by the troopers found that he was driving without a license and that he had an extensive criminal record in New York City.

On November 14, 1957, powerful mafiosi from the United States and Italy convened at Barbara's estate in Apalachin, New York. Cuba was one of the Apalachin topics of discussion, particularly the gambling and narcotics smuggling interests of La Cosa Nostra on the island. The international narcotics trade was also an important topic on the Apalachin agenda. The New York garment industry interests and rackets, such as loansharking to the business owners and control of garment center trucking, were other important topics on the Apalachin agenda.

In the time preceding the November 1957 meeting, trooper Croswell had Barbara's house under occasional surveillance. He had become aware that Barbara's son was reserving rooms in local hotels along with the delivery of a large quantity of meat from a local butcher to the Barbara home. That made Croswell suspicious, and he therefore decided to keep an eye on Barbara's house. When the state police found many luxury cars parked at Barbara's home they began taking down license plate numbers. Having found that many of these cars were registered to known criminals, state police reinforcements came to the scene and began to set up a roadblock.

Having barely started their meeting, Bartolo Guccia, a Castellammare del Golfo native and Barbara employee, spotted a police roadblock while leaving Barbara's estate. Guccia later said he was returning to the Barbara home to check on a fish order. Some attendees attempted to drive away but were stopped by the roadblock. Others trudged through the fields and woods ruining their expensive suits before they were caught. Many Mafiosi escaped through the woods surrounding the Barbara estate.

In 1957, after taking control of the Luciano crime family from boss Frank Costello, boss Vito Genovese wanted to legitimize his new power by holding a national Cosa Nostra meeting. Genovese elected Buffalo, New York boss and Commission member, Stefano "The Undertaker" Magaddino, who in turn chose northeastern Pennsylvania crime boss Joseph Barbara and Bufalino to oversee all the arrangements.

Following Barbara's death in June 1959, The Commission recognized Bufalino as the official family boss.

On April 20, 1973, Bufalino was arrested in a Scranton night club in an FBI raid, charged with interference with interstate commerce, obstruction of justice, gambling and transporting stolen property, but later released in $50,000 bail.

The police stopped a car driven by Bufalino, whose passengers included Genovese and three other men, at a roadblock as they left the estate; Bufalino said that he had come to visit his sick friend, Barbara. All those apprehended were fined, up to $10,000 each, and given prison sentences ranging from three to five years.

In 1977, Bufalino was indicted on extortion charges after Jack Napoli, who was in the Witness Protection Program, testified that Bufalino had threatened to kill him for failing to pay a $25,000 debt to a jeweler in New York. As soon as Bufalino was indicted, he took steps to reduce the possibility of further criminal charges. He named caporegime Edward Sciandra as acting boss and removed himself from the day-to-day operations of the family.

On August 8, 1978, Bufalino was convicted and sentenced to four years imprisonment for his part in the extortion attempt. He served almost three years.

Bufalino was released in May 1981, but was indicted again, this time for conspiring to kill the witness, Napoli. The main prosecution witness, Jimmy Fratianno, said that he and Michael Rizzitello had been asked by Bufalino to kill Napoli in 1976.

In November 1981, Bufalino was sentenced to 10 years imprisonment, held at United States Penitentiary, Leavenworth.

With Bufalino again in prison and the family under federal investigation, the organization's strength began to wane. In 1989, Bufalino was released from prison, and the operations of the remainder of the Northeastern family was given to Billy D'Elia.

On February 25, 1994, Bufalino died of natural causes at Nesbitt Memorial Hospital in Kingston, Pennsylvania, aged 90. He is buried in Denison Cemetery in Swoyersville, Pennsylvania.

Bufalino is portrayed by Joe Pesci in the 2019 Martin Scorsese film The Irishman.