Mazar began her career as a hip hop b-girl in New York City. Her first television appearance was on the pilot for a hip hop television dance show, Graffiti Rock in 1984. She appeared in five of Madonna's music videos – "Papa Don't Preach", "True Blue" (both 1986), "Justify My Love" (1990), "Deeper and Deeper" (1992) and "Music" (2000).

Mazar has played a number of minor supporting roles in a variety of films, including Sandy, a friend of Henry Hill's mistress in Goodfellas (1990); The Doors (1991); a small role in Spike Lee's Malcolm X (1992); Bullets Over Broadway (1994); and as Spice (of Sugar and Spice, with Drew Barrymore as Sugar) in Batman Forever (1995). She gained her first real following from playing a character on Civil Wars in the early 1990s. When that series was cancelled her character was brought over as a recurring role between 1993 and 1994 season of the TV drama L.A. Law.

Prior to her marriage, she dated actor Paul Reubens for several years beginning in 1993. Reubens has since credited Mazar with ending his depression resulting from his infamous 1991 arrest.

Mazar appeared on a Friends episode in its eighth season ("The One Where Rachel Has a Baby, Part One"). Mazar played "Doreen, the Evil Bitch," a crazed pregnant woman who shares a hospital room with Rachel. In the 1999 docudrama film The Insider she played character Lowell Bergman's assistant Debi. From 2000-02 she played Jackie on the television drama That's Life. She provided the voice of Maria Latore in the video games Grand Theft Auto III (2001) and Grand Theft Auto: San Andreas (2004).

She married Gabriele Corcos on March 16, 2002, in a ceremony officiated by Ellen Burstyn. They have two daughters, Giulia and Evelina. The family formerly lived in Los Angeles but moved to Brooklyn, New York in 2009. They opened a cafe/restaurant called Tuscan Gun. The family currently divides their time between Brooklyn and a 15th-century home outside of Florence, Italy, that was given to them as a wedding present by Mazar's in-laws.

From 2004-11, she had a supporting role on Entourage as press agent Shauna Roberts. She also had a recurring role on the sitcom Living with Fran, playing Fran Drescher's character's cousin, Merrill. She did a two-episode stint on the television series Ugly Betty as fraudster Leah Stillman.

Mazar was a contestant on the ninth season of Dancing With the Stars. She was partnered with Maksim Chmerkovskiy and finished in twelfth place. She was eliminated in the third week (October 6, 2009). In 2012, Mazar played Jessica, a glamorous, leather-clad villainess in Home Alone: The Holiday Heist.

Mazar and Corcos formerly hosted an internet show focused on Tuscan cuisine, Under The Tuscan Gun. On January 19, 2011, Debi, and her husband Gabriele began hosting the cooking show Extra Virgin on the Cooking Channel, and released a cookbook, Recipes & Love From Our Tuscan Kitchen, in 2014.

Mazar and her husband both appeared on an episode of the ABC talk show The Chew in 2014 to promote their show, Extra Virgin.

Beginning in 2015, Mazar has starred in Younger with Sutton Foster and Hilary Duff as Maggie. The series met critical acclaim and began filming its fifth season in February 2018.

Mazar appeared in the 2nd season of The $100,000 Pyramid reboot on ABC on August 6, 2017. In the main game, she helped her contestant get 7 clues in only 15 seconds causing host Michael Strahan to say, "I think that's the quickest round we've ever had, 15 seconds!"