In 2002, he had a brief appearance as one of the first Human-Form Replicators in the season 6 episode of Stargate SG-1, "Unnatural Selection". From 2004-2005, Penikett portrayed Ray Chase, who appeared in eleven episodes of the Canadian police drama, Cold Squad. He played a leading role as Noah Hamilton in the 2005 made-for-TV film Hush alongside Actress Tori Spelling. He had a role in the video game Need for Speed: Carbon, as a street racer named Darius. He also appeared in four episodes of the television series Whistler as Elias Noth. Penikett also provided voice talent for antagonist Troy Hammerschmidt on the Adult Swim show Titan Maximum. In 2010, Penikett played the lead role of Matt Ellman on Syfy's miniseries Riverworld an adaption of the science fiction books written by Philip José Farmer of the same name. He starred in the first two episodes of Warner Brothers' Mortal Kombat: Legacy as Kurtis Stryker, which debuted in April on YouTube; he was replaced by Eric Jacobus for season 2.

In 2003, the 1978 science fiction television series Battlestar Galactica was "reimagined" as a three-hour miniseries on the SciFi Channel. The miniseries was filmed in Vancouver, British Columbia, Canada, and Penikett, a local actor, auditioned and was cast in the part of Karl "Helo" Agathon, an officer in the Colonial Fleet. The miniseries proved highly successful, and a Battlestar Galactica series was commissioned.

Among his early acting work were stints on assorted Canadian TV series such as Cold Squad. He also appeared on the TV show Smallville, in the episode "Resurrection" in Season 3 as Vince Davis, and in the Season 6 episodes "Nemesis" and "Prototype" as Sgt. Wes Keenan. He portrayed a police officer for a predominantly gay neighbourhood in the 2004 The L Word episode "Losing It", which starred Battlestar Galactica co-star Nicki Clyne in a related storyline.

In support of the Babz Chula Lifeline for Artists Society, which is raising money for their friend and fellow Canadian actor Chula's cancer treatment, Penikett and his Battlestar Galactica colleague Kandyse McClure auctioned off the opportunity to have dinner with both of them in Vancouver in April 2008.

In 2009, Penikett was cast as Paul Ballard in Joss Whedon's science fiction drama television series Dollhouse, which aired on Fox network Friday nights at 9:00. During the first season, Ballard was the purported "good guy" cop who was trying to bring the Dollhouse down; however, an unhealthy fixation on Echo (the main character, portrayed by Eliza Dushku), as well as other factors, led to his expulsion from the FBI. In an attempt to both save Echo and himself, and in exchange for releasing former lover November (Miracle Laurie), Ballard joined the Dollhouse as Echo's handler during the second season, and worked insidiously from the inside in an attempt to set the dolls free from what he perceives as slavery and to bring down not just the Dollhouse but the power-hungry Rossum Corporation behind it.

Dollhouse was canceled at the end of its second season, and the series finale aired on Friday, January 29, 2010. In 2012, he guest-starred as a Politician named Jim Martin in the first season of Showcase's Continuum. He returned as a recurring character in the second season of the show. In 2013, Penikett was cast on the CW TV series Supernatural as a fallen angel who claimed to be Ezekiel, appearing in the show's ninth season premiere. The character's real name was revealed as Gadreel in episode nine and Penikett reprised the role in episode ten. He has since reprised the roles in episodes eighteen, twenty-one and twenty-two. He also landed another recurring role on the mid-season drama Star-Crossed, which premiered in February 2014.

In 2014, Penikett guest-starred in the 200th episode of Criminal Minds as Michael Hastings, an ex-CIA agent and leader of the terrorist group, The Regime Squad. Since 2014, he has also starred in the award-winning web series Riftworld Chronicles.