Kricfalusi says he is mostly self-taught, having only spent a year in Sheridan College, barely attending class. He acquired his skills largely by copying cartoons from newspapers and comic books as a child, and by studying cartoons and their production systems from the 1940s and 1950s. His main influence is Bob Clampett, and he also names Chuck Jones, Frank Sinatra, Kirk Douglas, Milt Gross, Tex Avery, Peter Lorre, The Three Stooges, Al Jolson, Bing Crosby, Dean Martin, Elvis Presley, Don Martin and Robert Ryan. Michael Barrier, an animation Historian, said that Kricfalusi's works "testify to his intense admiration for Bob Clampett's Warner Bros. cartoons" and that no Cartoonist since Clampett created cartoons in which the emotions of the characters "distort their bodies so powerfully".

After moving to Los Angeles, Kricfalusi was introduced to Milt Gray by Bob Clampett, suggesting he should join Gray's classical animation class. Gray was working for Filmation at the time, and soon Kricfalusi found work there as well, getting his start on the shows like Super Friends and The Tom and Jerry Comedy Show. His first independent cartoon was a short called Ted Bakes One, which he produced with Bill Wray in 1979 for a cable channel. From 1979 to the mid-1980s, Kricfalusi worked for Filmation and later Hanna-Barbera and DIC Entertainment on various shows that he once described as "the worst animation of all time". However, he did enjoy his work as a layout Artist on the 1985 series of The Jetsons as he was able to train a team of Taiwanese animators to draw characters more emotive and wild, which at the time was considered radical. He recalls being "saved" from having to work on these cartoons by Director Ralph Bakshi, who'd worked with him before in 1981 and 1982. They began working on the designs for the film Bobby's Girl, which was sold to Tri-Star but later cancelled. Under Bakshi, Kricfalusi directed the animation for The Rolling Stones' 1986 music video "Harlem Shuffle".

Kricfalusi formed Spümcø animation studio with partners Jim Smith, Bob Camp and Lynne Naylor. They began working on a pilot for The Ren & Stimpy Show on behalf of Nickelodeon, after the eponymous characters were favored by Nickelodeon Producer Vanessa Coffey in a presentation by Kricfalusi. The pilot was very well received, leading to the production of the first 13 half-hour episodes of the show. The show came to garner high ratings for Nickelodeon, and at the time was the most popular cable TV show in the United States, but the network disagreed with Kricfalusi's direction of the show, and disapproved of his missed production deadlines. Kricfalusi points specifically to the episode "Man's Best Friend", which depicts the character George Liquor as an abusive father figure, as the turning point in his relationship with Nickelodeon. One of the episodes, "Nurse Stimpy", did not meet Kricfalusi's approval because of the low quality of the rough cut of the episode that they received from the overseas studio, leading him to use the alias Raymond Spum in its credits. Nickelodeon terminated Kricfalusi's contract late September 1992, leaving it to Nickelodeon's Games Animation studio, which continued producing it for three more seasons before its cancellation.

After leaving The Ren & Stimpy Show, Kricfalusi consulted, and other Spümcø animators worked for Donovan Cook's 2 Stupid Dogs, which was put into production by Hanna-Barbera President Fred Seibert. The cartoon's credits read "Tidbits of Poor Taste Supplied by John Kricfalusi" for the three "Little Red Riding Hood" episodes: "Red!", "The Return of Red" and "Red Strikes Back". In 1994, Hanna-Barbera and Seibert started production on What A Cartoon!, also known as World Premiere Toons for Cartoon Network. Siebert approached Kricfalusi for advice and for recommendations for personnel to head the shorts, among them David Feiss, Tom Minton, and Eddie Fitzgerald.

Venturing into Internet cartoons, Kricfalusi created Weekend Pussy Hunt in 1996 for MSN, which was billed as "the world's first interactive web-based cartoon". The cartoon, which was released in segments, was scheduled to be completed on June 1997, but production under MSN stopped before it was finished. Production later resumed under Icebox.com after the release of Spümcø's own web-based Flash cartoon, The Goddamn George Liquor Program. Between 1998 and 2001 Kricfalusi worked on several Hanna-Barbera cartoons for Cartoon Network: three Yogi Bear cartoons he directed and animated, Boo Boo and the Man, A Day in the Life of Ranger Smith and Boo Boo Runs Wild, and two Jetsons cartoons he produced, The Jetsons: Father & Son Day and The Jetsons: The Best Son.

Fox Kids started airing the TV series The Ripping Friends in 2001, created by Kricfalusi and Jim Smith. Kricfalusi had previously tried pitching the show in the late 80's, but networks considered it "too extreme" so did not pick it up. Kricfalusi felt the show's supervisors were doing away with the Spümcø style, and was displeased with the direction of the show. He was not fully involved in the show until half-way through production and considers the episodes he was involved in to be experimental. One of his contributions to the show was directing the voice-actors, which he "really worked-out" so much that he was afraid he'd give one of them a heart-attack.

In 2003, Spike TV produced a new show featuring Ren & Stimpy, which was written and directed by Kricfalusi. The first three episodes were based on fan ideas and scripts that were rejected by Nickelodeon during the original show's run. According to Kricfalusi, Spike pushed for more South Park-like themes in the new show. While he was initially pleased with the added freedom afforded to him by Spike, he later expressed disappointment in the series due to its slow pacing and overuse of toilet humor. Only three episodes aired before Spike's entire animation block was "put on hold", and the complete series was ultimately released in 2006 on DVD including three additional episodes that never aired. Kricfalusi also wanted to release an episode titled "Life Sucks" straight to DVD, but the episode remains unproduced.

Kricfalusi contributed several articles in 1993 and 1994 for the magazines Film Threat and Wild Cartoon Kingdom under various aliases. Kricfalusi appears in several bonus featurettes and provides audio commentaries for the Looney Tunes Golden Collection volumes 2, 3 and 5, for cartoons directed by Bob Clampett and Chuck Jones. On February 13, 2006, Kricfalusi started his own weblog, John K Stuff, posting about cartoons and the animation industry. The site was originally intended for other artists and entertainers, and specifically other cartoonists.

Kricfalusi directed commercials for Comcast and Voice over IP company Raketu in 2007. He was developing a series of cartoon commercials in 2008 for Pontiac Vibe starring George Liquor and Jimmy The Idiot Boy, but the series remained unreleased after General Motors discontinued the Pontiac Vibe auto line in 2009. He developed and animated a series of bumpers using Toon Boom Harmony for Adult Swim in 2011 and again in 2015. He animated the opening couch gags of two episodes of The Simpsons, "Bart Stops to Smell the Roosevelts", which aired in October 2011 and "Treehouse of Horror XXVI", which aired in October 2015. He collaborated with streetwear brand Stüssy to create a short series of apparel based on his designs in 2012, which he promoted with a commercial featuring some of his characters. Also in 2012, he funded through Kickstarter a cartoon short entitled "Cans Without Labels" starring the character George Liquor, with the initial delivery date of February 2013. The cartoon was due to be screened at the 2016 Annecy International Animated Film Festival for the first time, however at the last minute it was announced that it wasn't ready. However, on August 6, 2017, the Kickstarter has been updated finally announcing the film's completion. The advertising agency Muhtayzik-Hoffer hired Kricfalusi in 2013 for an ad campaign for F'real milkshakes. He was involved in the early development of many Reel FX projects such as the 2013 film Free Birds, a pitch for a film adaptation of the Dr. Seuss book Happy Birthday to You! and a pitch for a film he created with Jim Smith. He posted the concepts for these projects on his blog. He partnered with Animator Mike Judge to produce a series of shorts for UFC that aired on Adult Swim throughout 2016.

Animators Robyn Byrd and Katie Rice alleged in a BuzzFeed report on March 29, 2018, that they were sexually harassed and groomed for sexual abuse by Kricfalusi while they were underage. Byrd said that she was in a sexual relationship with Kricfalusi in 1997, when she was 16, and moved in with him, in California, when she was 17. Rice said that she was sexually harassed by Kricfalusi when she turned 18 and began working at his animation studio, Spümcø. Documents they had saved from those years corroborate their stories, and several people who worked with Kricfalusi referred to his sexual harassment as an open secret in the animation industry. Kricfalusi was also alleged to possess child pornography on his computer. Kricfalusi's Lawyer confirmed that "for a brief time, 25 years ago, he had a 16-year-old girlfriend", but denied that Kricfalusi's "avid pursuit" of Rice was sexual harassment or that he had ever possessed child pornography.