Marie Louise Hartman was born in Berkeley, California, to a Lutheran father and a Jewish mother (born 1926) whose family was from Alabama. She grew up in the San Francisco Bay Area. She is the youngest of four children, with an older sister and two older brothers. Her parents were communists who converted to Buddhism when she was young. After graduating from Berkeley High School in 1977, she attended San Francisco State University's undergraduate nursing school and graduated magna cum laude in 1985. She was a registered nurse per the California Board of Registered Nursing until her license expired in 1986.

In the 1980s and early 1990s, she starred in several of the Debbie Does series, such as Debbie Does Dishes and Debbie Does Wall Street.

Hartley is openly bisexual in her private life. From 1981–2000, Hartley was involved in a relationship with a man and a woman. She married her male partner in 1986; their divorce was finalized in 2003.

In 1982, during her sophomore year of nursing school, she started working as a stripper at the Sutter Cinema and later the Mitchell Brothers O'Farrell Theatre. She made her foray into the world of pornographic movies during her junior year in 1984. Her debut performance was in the film Educating Nina, making her a protégée of Juliet Anderson.

Hartley had a role in the 1997 film Boogie Nights, playing william H. Macy's serially unfaithful wife, and she appeared in the 1996 Canadian film Bubbles Galore. Hartley also appeared in the late Rapper Tupac Shakur's music video for his 1996 single, "How Do U Want It?"

In 2003, the year her divorce was finalized, she married Ernest Greene, professional name Ira Levine, a Director of porn films.

In 2006 she published her first book Nina Hartley's Guide to Total Sex (ivbro). She would later co-author the book series "How to be Kinkier" beginning in 2012.

In 2010, she quipped, "Now I work with women who are younger than my breast implants."

Hartley was also featured in the 2012 documentary After Porn Ends, which is about life after being a porn actor.

Nina Hartley appears in the Amazon Award winning 2016 documentary Sticky: A (Self) Love Story, in which she discusses her sex positive views on self-love, with regards to education, the forced resignation of Joycelyn Elders, and her opinions on the blackballing of Comedian Paul Reubens after his arrest for masturbating in a public theater. Nina Hartley attended a Las Vegas premiere of the documentary at the Erotic Heritage Museum on May 26, 2016, in which she sat on a Q&A panel with the writer/producer/director Nicholas Tana, and adult film Actress Ginger Lynn.

As of 2017, Hartley remained active in the industry. Appearing primarily in "mature"-themed pornography, in 1994 she began her line of instructional videos which are marketed under the Nina Hartley's Guide brand covering subjects ranging from basic sexual intercourse and foreplay, to anal sex and bondage.