He graduated from william Penn Charter School in 1971 and originally entered Yale University as a pre-medical student, but opted to act instead. He ended up graduating with a bachelor's degree in drama from Yale University.

After earning his degree, he enrolled at the Circle in the Square Professional Theater Workshop. He waited tables for a few years until his theatrical work started to take off around 1976. His first breaks were appearing in the David Mamet play “Sexual Perversity in Chicago,” and with Diane Keaton in “The Primary English Class”.

Picardo appeared on Kojak in a 1977 episode, E-Ring, as a media rep in The Pentagon, as an enraged father in Cold Case, and as a police officer in CSI: NY. He was a recurring guest star in two episodes of Season 7 of Smallville.

Picardo made his feature film debut as Eddie Quist, the serial killer werewolf in the Joe Dante film The Howling (1981). He also had a recurring role in the sitcom Alice and played a Doctor on an episode of The Golden Girls. He played a number of roles in Dante's family science fiction film Explorers (1985), and later appeared in Dante's The 'Burbs, Looney Tunes: Back in Action, Matinee, Gremlins 2: The New Batch, Small Soldiers and Innerspace. Picardo often plays roles under layers of prosthetic latex, having also played the swamp-dwelling Meg Mucklebones in Ridley Scott's film Legend. He also had a small role as a funeral Director in John Landis's Amazon Women on the Moon. He voices Pfish in two Pfish & Chip shorts as seen on Cartoon Network's What-A-Cartoon! Show. Picardo also portrays the voice and face of the robotic Johnny Cab in Total Recall. He appeared in one episode of ER in 1995 as Abraham Zimble (Season 2 - Episode 6, "Days Like This").

During the 1988-1991 television seasons, Picardo was simultaneously seen on the ABC Vietnam series China Beach in the role of Dr. Dick Richard, and the ABC series The Wonder Years in the role of Coach Cutlip. He is among a small group of television actors to achieve notice on two television series at the same time.

In the early 1990s, Picardo had a brief role as Joe "The Meat Man" Morton, a butcher and neighbor to Tim Allen's character on the sitcom Home Improvement.

From 1995 to 2001, he played the role of the Emergency Medical Hologram (EMH) in the television series Star Trek: Voyager. Before being accepted for this role, Picardo initially auditioned for the role of Neelix. He later also directed two episodes.

He played additional versions of the role of the EMH in the 1996 motion picture Star Trek: First Contact and the 1997 Star Trek: Deep Space Nine episode "Doctor Bashir, I Presume?" He also played Dr. Lewis Zimmerman, the creator of the EMH, in episodes of Deep Space Nine and Voyager.

Away from acting, from 1999-2015, Picardo was a member of the Board of Directors' Advisory Council of The Planetary Society; beginning in 2015, he was elected to the Board of Directors itself.

In 2001, he guest starred in the 7 Days episode "Revelation", purporting to be a time traveler from seven years in the Future.

In 2002, Picardo authored The Hologram's Handbook, published by Pocket Books.

In 2004, he began playing the recurring role of International Oversight Advisory (IOA) member Richard Woolsey in both Stargate SG-1 and Stargate Atlantis. His first appearance in those series was in the Stargate SG-1 episode "Heroes (Part 2)".

In 2007 and 2008, Star Trek: The Music was a multi-city tour with John de Lancie. Picardo and de Lancie narrated around the orchestral performance, explaining the history of the music in Star Trek.

On February 5, 2008, it was announced that Picardo would be joining the regular cast of Stargate Atlantis full-time for the series' fifth and final season. He took over the role of mission commander of the Atlantis Expedition.

Picardo performed the voice of Loki in the Xbox 360 video game Too Human. In 2009, he also appeared in Pushing Daisies, Chuck and Castle. Also in 2009, he played the lead role in the independent psychological thriller film Sensored. In 2010, Picardo had a cameo in the final episode of Persons Unknown, as a member of "the program"'s governing board. Picardo also voices Robert McNamara in Call of Duty: Black Ops in campaign and in the 'Zombie mode' after completing campaign on the one map, "FIVE". Picardo appeared in four episodes of The Mentalist from 2012 through 2013 as Jason Cooper, a lieutenant of cult leader Bret Styles.

In May 2014, Cartoon Hangover announced Robert Picardo as a guest voice actor in the second season of Bravest Warriors, in the episode "The Parasox Pub".

In 2015, Picardo reprised the role of Dr. Lewis Zimmerman in the pilot episode of the fan series Star Trek: Renegades

In 2017, Robert Picardo appeared as a guest actor, playing the father of Lt. Kitan, in the first season of The Orville, in the episode "Firestorm". Picardo will reprise the role in season 2.