Although showing no interest in acting as a child, save a brief appearance on Sesame Street, Rosario was discovered on her front porch step by photographer Larry Clark and Harmony Korine, where Harmony lauded her with praise as being perfect for a part he had written in his screenplay that would become the controversial 1995 film Kids. Since then Dawson's films have varied; ranging from independent films, to highly successful big budget blockbusters, and large scale box office bombs. Among her successes are Rent, He Got Game and Men in Black II. Among her failures are The Adventures of Pluto Nash (which was nominated for six Golden Raspberry Awards) and the live-action film adaptation of Josie and the Pussycats. In 1999, Dawson teamed up with Prince for the re-release of his 1980s hit "1999". The new remixed version featured the actress in an introductory voice over, offering commentary on the state of the world in the year before the Millennium. The same year she appeared in The Chemical Brothers' video for the song "Out of Control" from the album Surrender. She is also featured on the track "She Lives In My Lap" from the second disc of the OutKast album Speakerboxxx/The Love Below, during which she speaks the intro and a brief interlude towards the end.

In 2004, Dawson appeared in Oliver Stone's Alexander as the bride of Alexander the Great, which also featured her in a fully nude scene. In August-September 2005, Dawson appeared on stage as Julia in the Public Theatre revival of Galt MacDermot's 1971 musical Two Gentlemen of Verona at the Delacorte Theater in Central Park. She starred in the film adaptation of the popular musical Rent, where she played the exotic dancer Mimi Marquez, replacing the original Mimi, Daphne Rubin-Vega, who was pregnant at the time of filming and wasn't able to play the part. She also appeared in the adaptation of the graphic novel Sin City, where she played the prostitute-dominatrix, Gail. She will appear in Sin City's sequel in 2008.

In 2005, Dawson appeared in a graphically violent scene in the Rob Zombie film The Devil's Rejects. Though the scene was cut from the final film, it is available in the deleted scenes on the DVD release. In 2006's Clerks II, Dawson starred as Becky, the crush-turned-wife of Dante Hicks. As she mentioned in the making of documentary, Back to the Well, the donkey show sequence was what made her decide to appear in the movie. In May of the same year, Dawson, an avid comic book fan, co-created the comic book miniseries Occult Crimes Taskforce. She was at the 2007 Comic-Con to promote her new comic book miniseries. In 2007, Dawson co-starred with former Rent alum Tracie Thoms in the Quentin Tarantino and Robert Rodriguez's throwback movie Grind House. In 2007, Dawson teamed up with friend Talia Lugacy, whom she met at the Lee Strasberg Academy, to produce and star in Descent. On July 7, 2007, Dawson presented at the American leg of Live Earth. On June 26, 2008, it was announced that Dawson will play Artemis in the upcoming animated Wonder Woman film. Starting on August 18, 2008, Dawson starred in Gemini Division, an online-based TV series. In 2008, Dawson starred with Will Smith in Seven Pounds. She played the dying Emily Posa who's heart was failing. The movie got critical reception from most critics, but IMDB rated it a 7.5 out of 10 with 7639 as of December 29, 2008.

• Poor Things (2008) (in production)
• Sin City 2 (2010) (pre-production) .... Gail
• Wonder Woman (2009) (V) (completed) (voice) .... Artemis
• The Haunted World of El Superbeasto (2009) (completed) (voice) .... Velvet Von Black
  
• Seven Pounds (2008) .... Emily Posa
• Killshot (2008) .... Donna
• "Gemini Division" .... Anna Diaz (30 episodes, 2008)
       - Bound (2008) TV episode .... Anna Diaz
       - Laszlo (2008) TV episode .... Anna Diaz
       - Training Day (2008) TV episode .... Anna Diaz
       - Smart Cows (2008) TV episode .... Anna Diaz
       - La Mujer Dormida (2008) TV episode .... Anna Diaz
        (25 more)
• Eagle Eye (2008) .... Zoe Perez
  ... aka Eagle Eye - Außer Kontrolle (Germany)
  ... aka Eagle Eye: The IMAX Experience (USA: IMAX version)
• Explicit Ills (2008) .... Babo's Mom
• "Robot Chicken" .... Clara Palmer / ... (1 episode, 2007)
       - More Blood, More Chocolate (2007) TV episode (voice) .... Clara Palmer/Woman
• Death Proof (2007) .... Abernathy
  ... aka Grindhouse Presents: Quentin Tarantino's Death Proof (USA: DVD title)
  ... aka Grindhouse: Death Proof (International: English title)
  ... aka Quentin Tarantino's Death Proof (USA: complete title)
  ... aka Quentin Tarantino's Thunder Bolt (USA: informal alternative title)
• Descent (2007) .... Maya
• Grindhouse (2007) .... Abernathy (segment "Death Proof")
• A Guide to Recognizing Your Saints (2006) .... Laurie
• Clerks II (2006) .... Becky
• Rent (2005) .... Mimi Marquez
• Little Black Dress (2005) .... Haley
• The Devil's Rejects (2005) (scenes deleted) .... Nurse Marcia
  ... aka TDR - The Devil's Rejects (Germany)