Garofalo's big break came in 1990 after meeting Ben Stiller at Canter's Deli in Los Angeles, where they were hanging out with stand-up friends. They bonded over their "love of SCTV, early Saturday Night Live, and Albert Brooks."

Garofalo's first movie role, filmed the year before she appeared on national television, was a brief comical appearance as a counter worker in a burger joint in Late for Dinner in 1991, but her real breakthrough into film came in Reality Bites (1994) as Winona Ryder's character's Gap-managing best friend Vickie. The role helped solidify Garofalo's status as a Generation X icon.

Her first exposure on national television came soon thereafter by way of her appearance as a stand-up comic on MTV's Half Hour Comedy Hour. Subsequently, her first television series debut was on the short-lived Ben Stiller Show on Fox in 1992, on which she was a cast member alongside longtime friends Bob Odenkirk and Andy Dick.

After The Ben Stiller Show was cancelled, Garofalo joined the cast of Saturday Night Live (SNL) for its 1994–95 season. She left SNL in March 1995 (mid-season) after only six months, saying that the experience left her "anxious and depressed" and that a sexist attitude pervaded the show and she called many of the sketches "juvenile and homophobic". According to New York Magazine, Garofalo was "largely stuck in dull, secondary wife and girlfriend roles" and her friends said that she considered the stint "the most miserable experience of [her] life."

She did an HBO Comedy Half-Hour special in 1995, among similar subsequent appearances, including a one-hour stand-up special in June 2010 entitled "If You Will," performed at Seattle's Moore Theatre, that aired on Epix in June 2010 and was released on DVD in September 2010.

A chance meeting on the set of that show led her to being offered the role of Paula on The Larry Sanders Show on HBO, earning her two Primetime Emmy Award for Outstanding Supporting Actress in a Comedy Series nominations in 1996 and 1997. For a time, she was actually working on both series during the same period.

Garofalo co-wrote a comedic New York Times bestseller with Ben Stiller in 1999, entitled Feel This Book: An Essential Guide to Self-Empowerment, Spiritual Supremacy, and Sexual Satisfaction, a spoof of the self-help books so prevalent at the time. She wrote her HBO Comedy Half-Hour along with similar appearances and programs, co-wrote some sketches on The Ben Stiller Show, wrote an episode of the television series Head Case, and wrote and directed a 2001 comedy short entitled Housekeeping.

She has appeared with political figures such as Ralph Nader (whom she supported in the 2000 election, but opposed in 2004) and Jello Biafra at various events. In 2007, Garofalo described herself as an atheist, and participated in a radio interview by Freethought Radio, a show by the Freedom From Religion Foundation.

In 2002, she played Catherine Connolly in The Laramie Project and in 2003, she starred in Manhood and Ash Tuesday, and appeared in the crime film Wonderland. She played a supporting role in Jiminy Glick in Lalawood in 2004.

In March 2003, she took part in the Code Pink anti-war march in Washington, D.C. That autumn, she served as emcee at several stops on the Tell Us the Truth tour, a political-themed concert series featuring Steve Earle, Billy Bragg, Tom Morello, and others. Throughout the year, Garofalo also actively campaigned for Howard Dean. While on Fox News' program The Pulse, O'Reilly asked Garofalo what she would do if her predictions that the Iraq war would be a disaster were to turn out wrong. Garofalo stated:

In late March 2004, Garofalo became a co-host for Air America Radio's new show The Majority Report, alongside Sam Seder. The early days of Air America Radio are chronicled in the documentary Left of the Dial, which includes a debate between Garofalo and her conservative Father Carmine, who was initially a regular guest on The Majority Report.

Throughout the 2005–06 television season, Garofalo appeared on The West Wing as Louise Thornton, a controversial campaign adviser to the fictional Democratic presidential nominee. Garofalo participated in the series' first live episode, most of which was a debate televised live on the East Coast and then reshot live for the West. Garofalo's character can be seen walking backstage advising before the start of each debate. In 2006, she provided the voice for the animated character "Bearded Clam" on Comedy Central's Freak Show. In 2007, she wrote a dedication for the mini-book included in the six-DVD box-set of the 1994 cult series My So-Called Life.

Garofalo commented on her show of April 28, 2006 supporting the Scientology-linked New York Rescue Workers Detoxification Project, a controversial treatment for workers suffering ailments from 9/11 clean-up efforts in New York City.

Garofalo said she had misgivings in 2007 about the depiction of torture in the television series 24 but joined the cast because "being unemployed and being flattered that someone wanted to work with me outweighed my stance".

In April 2009, Garofalo drew criticism from The Washington Times when she denounced Tea Party protests, which she referred to as racist. She has continued to criticize Tea Party protesters.

In Garofalo's 2010 stand-up show, If You Will, she says "I don't have a fear of intimacy, I have sort of a genuine lack of interest", adding jokingly, "which is not good for my boyfriend of ten years".

In 2014, she portrayed Lyla, an entertainment Lawyer, in seven episodes of the TV series Girlfriends' Guide to Divorce. In 2015, she starred alongside most of the original cast in the Netflix eight-episode prequel to the 2001 comedy film Wet Hot American Summer. In 2017, Garofalo starred in E4's comedy-drama series Gap Year.

Janeane Garofalo appears as herself in the documentary film Sticky: A (Self) Love Story, released on Amazon in 2016, in which she claims that she had to get drunk on tequila to do her masturbation scene in the film Truth About Cats and Dogs.