During high school, Barbera worked as a tailor's delivery boy. In 1929, he became interested in animation after watching a screening of Walt Disney's The Skeleton Dance.  During the Great Depression, he tried unsuccessfully to become a cartoonist for The NY Hits Magazine. He supported himself with a job at a bank and continued to pursue publication for his cartoons. His magazine drawings of single cartoons, not comic strips, began to be published in Redbook, The Saturday Evening Post, and Collier's—the magazine with which he had the most success.  Barbera also wrote to Walt Disney for advice on getting started in the animation industry. Disney wrote back, saying he would call Barbera during an upcoming trip to New York, but the call never occurred.

Barbera's desk was opposite that of William Hanna. The two quickly realized they would make a good team. By 1939, they had solidified a partnership that would last over 60 years. Barbera and Hanna worked alongside animator Tex Avery, who had created Daffy Duck and co-created Bugs Bunny for Warner Bros. and directed Droopy cartoons at MGM.  

In 1940, Hanna and Barbera jointly directed Puss Gets the Boot, which was nominated for an Academy Award for Best (Cartoon) Short Subject. The studio wanted a diversified cartoon portfolio, so despite the success of Puss Gets the Boot, Barbera and Hanna's supervisor, Fred Quimby, did not want to produce more cat and mouse cartoons, believing that those were already enough. Surprised by the success of Puss Gets the Boot, Barbera and Hanna ignored Quimby's resistance and continued developing the cat-and-mouse theme.

Modeled after the Puss Gets the Boot characters with slight differences, the series followed Jerry, the pesky rodent who continuously outwitted his feline foe, Tom.  Hanna said they settled on this cartoon's cat and mouse theme because "we knew we needed two characters". We thought we needed conflict, chase, and action. And a cat after a mouse seemed like a good, basic thought.  The revamped characters first appeared in 1941's The Midnight Snack. Over the next 17 years, Barbera and Hanna worked exclusively on Tom and Jerry, directing more than 114 popular cartoon shorts. During World War II, they also made animated training films.

In December 1966, Hanna-Barbera Productions was sold to Taft Broadcasting, renamed Great American Communications in 1987, for $12 million. Barbera and Hanna remained at the head of the company until 1991. The company was sold to the Turner Broadcasting System for an estimated $320 million. Turner began using Hanna-Barbera's television catalog as material for its new Cartoon Network cable channel in 1992, and by the mid-1990s, Hanna-Barbera was producing several original series for Cartoon Network, among them Dexter's Laboratory and The Powerpuff Girls. In 1996, Turner merged with Time Warner, owners of Warner Bros., who would eventually absorb Hanna-Barbera into Warner Bros. Animation.

Barbera and Hanna continued to advise their former company. They periodically worked on new Hanna-Barbera shows, including shorts for the series The Cartoon Cartoon Show and feature film versions of The Flintstones (1994) and Scooby-Doo (2002). In a new Tom and Jerry cartoon produced in 2000, The Mansion Cat, Barbera voiced the house owner.

Ten days before Hanna's death from throat cancer in March 2001, Hanna-Barbera was absorbed into Warner Bros. Animation, with the unit dedicated to the Cartoon Network original series spun off into Cartoon Network Studios. Barbera remained active as an executive producer for Warner Bros. on direct-to-video cartoon features and television series such as What's New, Scooby-Doo? and Tom and Jerry Tales. He also wrote, co-storyboarded, co-directed, and co-produced The Karate Guard (2005), the return of Tom and Jerry to the big screen. His final animated project was the direct-to-video feature Tom and Jerry: A Nutcracker Tale.