Dawson was born on May 9, 1979, in New York City. Her mother, Isabel Celeste, is of Cuban and Puerto Rican ancestry. Isabel was 16 years old when Rosario was born; she never married Rosario’s biological father, Patrick C. Harris. When Rosario was a year old, her mother married Greg Dawson, a construction worker. Dawson has a half-brother, Clay, who is four years younger.

In 1998, 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 new millennium. The following 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, in which she speaks the intro and a brief interlude towards the end.

In 2001, she appeared in the movie Josie and the Pussycats as band member Valerie Brown.

Dawson starred as Naturelle Rivera, the love interest of a convicted drug dealer played by Edward Norton, in the 2002 Spike Lee film drama, 25th Hour. In the 2004 Oliver Stone film Alexander, she played the bride of Alexander the Great. In the autumn of 2005, Dawson appeared on stage as Julia in the Public Theater’s “Shakespeare in the Park” revival of Two Gentlemen of Verona. It was her first appearance on stage.

Dawson was arrested in 2004, while protesting against president George W. Bush.

In the film adaptation of the popular musical Rent in 2005, she played the exotic dancer Mimi Marquez, replacing Daphne Rubin-Vega, who was pregnant and unable to play the part. She also appeared in the adaptation of the graphic novel Sin City, co-directed by Robert Rodriguez and Frank Miller, portraying Gail, a prostitute-dominatrix. Also in that year, she 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.

She starred as Becky in 2006’s Clerks II, and mentioned in Back to the Well, the making-of documentary, that the donkey show sequence was what made her decide to take the role. In May of the same year, Dawson, an avid comic book fan, co-created and co-wrote the comic book miniseries Occult Crimes Taskforce. She was at the 2007 San Diego Comic-Con to promote the comic. She co-starred with former Rent alum Tracie Thoms in the Quentin Tarantino throwback movie Death Proof in 2007, part of the Tarantino/Robert Rodriguez double feature Grindhouse. She 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.

In 2008, Dawson starred with Will Smith in Seven Pounds and in Eagle Eye, produced by Steven Spielberg. Beginning in August, she starred in Gemini Division, an online science fiction series. In the computer animated series Afterworld, she voiced the character Officer Delondre Baines. On January 17, 2009, Dawson hosted Saturday Night Live. Later in the year, she voiced Artemis of Bana-Mighdall in the animated film Wonder Woman.

In October 2008, Dawson became a spokeswoman for TripAdvisor.com’s philanthropy program, More Than Footprints, Conservation International, Doctors Without Borders, National Geographic Society, The Nature Conservancy and Save The Children. Also in October 2008, she lent her voice to the RESPECT! Campaign, a movement aimed at preventing domestic violence. She recorded a voice message for the Giverespect.org Web site stressing the importance of respect in helping stop domestic violence. In 2012, Dawson partnered with SodaStream International in launching the first annual Unbottle the World Day, a campaign conceived in an effort to raise awareness to the impact of cans and plastic bottles on the environment. Dawson also sits on the Board of Directors of Scenarios USA, which works to support a generation of reflective, outspoken, and confident youth through filmmaking and uses film to educate students through a variety of programs.

In 2009, Dawson performed in The People Speak, a documentary feature film that uses dramatic and musical performances of the letters, diaries, and speeches of everyday Americans, based on historian Howard Zinn’s A People’s History of the United States. In 2009, Dawson also voiced the character of Velvet Von Black in Rob Zombie’s animated feature, The Haunted World of El Superbeasto. For the Kasabian album West Ryder Pauper Lunatic Asylum, she is featured singing on the track “West Ryder Silver Bullet”.

In 2010, she starred in the movies Percy Jackson & the Olympians: The Lightning Thief, as Persephone, and Unstoppable, as railway yardmaster Connie. In 2013, she played Apple’s mother in the independent film Gimme Shelter. The following year, she reprised her role as Gail in Sin City: A Dame to Kill For. In 2015, she played Claire Temple in the Netflix web television series Daredevil, a role which she reprised in Jessica Jones and Luke Cage. Dawson’s likeness was also used in the Jessica Jones tie-in comic as her character on both shows. Dawson has continued this role in 2017 in Iron Fist and The Defenders. In 2018, she played the female lead role in the Netflix movie, Krystal. In 2020, she was cast as the Star Wars character Ahsoka Tano in the second season of The Mandalorian on Disney+.

Dawson endorsed Barack Obama for re-election in 2012, and Bernie Sanders for the Democratic nomination in the 2016 Democratic Party primaries. On April 15, 2016, Dawson was among the protesters arrested during Democracy Spring in Washington, D.C.

From 2016 to 2017, Dawson dated comedian and television host Eric Andre.

In March 2019, Dawson confirmed that she is in a relationship with United States senator Cory Booker.

In October 2019 Dedrek Finley, a trans man, filed a case in Los Angeles against Dawson and her family for alleged incidents involving discrimination, verbal abuse, misgendering and physical assault. Finley had been employed as a handyman, living with the family and had known them for decades. Through their attorney, the Dawson family said that they are “saddened and disappointed by these false and baseless allegations.”

In February 2020, Dawson publicly came out as a member of the LGBT community.

In mid-2019, Dawson endorsed her boyfriend Cory Booker in the 2020 presidential election. Booker ended his campaign for president on January 13, 2020. Had she become First Lady of the United States, Dawson said she would have advocated for solutions to youth homelessness. On March 9, 2020, Dawson endorsed the presidential campaign of Bernie Sanders, whom she had also previously endorsed in his 2016 bid.