Cardellini was born in Redwood City, California, the daughter of Lorraine (née Hernan) and Wayne David Cardellini. She made her first public appearance at age 10, when she sang in a school play. After the performance, she plunged into several school productions and started attending drama lessons.[1] She graduated from nearby Mountain View's St. Francis High School in 1993.

Cardellini received her first big break in 1996 when she landed a starring role as Sarah on ABC's Saturday morning live-action children's series, Bone Chillers. Following this, she made guest appearances on prime-time programs such as Step by Step, Clueless, 3rd Rock from the Sun, and Boy Meets World as Lauren, a girl that came between the show's star couple. Cardellini spent the summer of 1999 in Europe as part of a touring production of Lancelot, a 14th century Dutch tragedy

Cardellini had her first major success when she landed one of the starring roles in the NBC series Freaks and Geeks, which debuted during the 1999-2000 season. As Lindsay Weir, an honor student in the midst of an identity crisis, the actress earned positive response that subsequently catapulted her fame. In 2002, Cardellini starred in the live-action adaption of Scooby-Doo, in which she brought to life the cartoon character of Velma Dinkley. She would later reprise the role of Velma in 2004's Scooby-Doo 2: Monsters Unleashed. In 2003, she joined the cast of the hospital drama ER as Samantha Taggart, a free-spirited nurse.

Her other film work includes roles in Dead Man on Campus, Legally Blonde, Brokeback Mountain, and a starring role in Happy Madison's Grandma's Boy as Samantha. She was also the voice of Ursula in the role-playing video game, Gladius. She is currently entering her fifth season as Samantha on ER. In 2007, Cardellini was chosen to play lovelorn Clara in CBS' miniseries Comanche Moon, a prequel to 1989's Lonesome Dove.

Cardellini attended Loyola Marymount University's College of Communication and Fine Arts, graduating in 2001 with a degree in theatre arts. She was also an active member of the Delta Zeta sorority. Cardellini dated her Freaks and Geeks co-star Jason Segelfor a few years following the show's cancellation. She enjoys art (especially that of Margaret Keane) and takes martial arts classes.