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. He played a number of roles in Dante's 1985 family science fiction film Explorers, and later appeared in Dante's The 'Burbs, Looney Tunes: Back in Action, Matinee, Gremlins 2: The New Batch, Small Soldiers, and Innerspace, as the Cowboy. 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 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").
In the early 1990s, Picardo had a recurring role as Joe "The Meat Man" Morton, a butcher and neighbor to Tim Allen's character on the popular sitcom Home Improvement. From 1995 to 2001, he played the role of the Emergency Medical Hologram (EMH) and Emergency Command Hologram (ECH) in the TV series Star Trek: Voyager. He also played additional versions of the role of the EMH in the 1996 Star Trek motion picture Star Trek: First Contact and the 1997 Star Trek: Deep Space Nine episode "Doctor Bashir I Presume?". In 2001, he guest starred in 7 Days episode "Revelation", purporting to be a time traveler from 7 years in the future. In 2004, he began playing the recurring role of International Oversight Advisory (IOA) member Richard Woolsey in both Stargate SG-1 and Stargate Atlantis, where he became a regular in the fifth season. His first appearance in those series was in "Heroes (Part 2)".