2000–2019: Spy Kids and Machete films
After concluding Animal Factory in 1999, he contracted Hepatitis C and "had to drag [his] ass" from Canada to Austin, Texas, to begin filming of Spy Kids in 2000. Spy Kids marked Trejo's debut as the fictional character Isador "Machete" Cortez. Having already made Desperado and From Dusk till Dawn together, the opportunity to collaborate with Robert Rodriguez, Antonio Banderas, and Cheech Marin once again "felt like [a] family reunion." Spy Kids provided Trejo with worldwide recognition and for the first time he was "instantly recognizable" among children around the globe.
By the time of Bubble Boy in 2001, his illness had progressed to the point that much of the cast had noticed his weight loss; Trejo states that his past drug use had caught up with him. He described himself as having been pale and weak throughout production, and pre-occupied with keeping his diagnosis a secret within Hollywood for fear of reprisal. Trejo was "out of it" and struggling to remember his lines due to prescription medication. By the time Spy Kids premiered in September 2002, Trejo had fully recovered.
Throughout the 2000s, Trejo appeared in productions including: XXX; Once Upon a Time in Mexico; Anchorman: The Legend of Ron Burgundy; The Devil's Rejects; Snoop Dogg's Hood of Horror; Delta Farce; Grindhouse; Rob Zombie's Halloween; Urban Justice (alongside Steven Seagal); and Valley of Angels. He also made a number of television appearances, including: Monk, Desperate Housewives, Stargate: Atlantis, and Breaking Bad. Trejo also voiced Enrique and several other minor characters on King of the Hill. His life is documented in the independent biographical film Champion, featuring some of Trejo's close friends: Dennis Hopper, Val Kilmer, Steve Buscemi, and Robert Rodriguez. Trejo also shared his tumultuous journey from convict to film star with KTTV in Los Angeles, 2013, in a segment filmed in Trejo's home.
Regarding his continued growth as a professional actor, Trejo has remarked, "I'm so blessed. I'm still scared that somebody's going to wake me up and say, 'Hey, we're still in prison. Let's go to chow'". Trejo also played 'Machete' in a trailer made for Rodriguez's film collaboration with Quentin Tarantino, Grindhouse. He also starred in a full theatrical release of the film Machete, in 2010, based on the character Isador "Machete" Cortez and again in 2013 for the sequel film, Machete Kills.
In 2011, he appeared in the action film Recoil as Drayke Selgado, with WWE wrestler and actor Steve Austin and played the role of the Ripper in Cross. In 2012, Trejo starred alongside Ron Perlman and Charles S. Dutton in the Craig Moss action film Bad Ass. He played the main character of Vietnam veteran Frank Vega, based on 67-year-old "Epic Beard Man" Thomas Bruso. That same year, Trejo appeared again with Ron Perlman, in a supporting role as Romero 'Romeo' Parada on season four of the FX television drama Sons of Anarchy. In 2014, Trejo produced his first film, titled Ambition, and produced his second film, the action film Bad Asses.
In 2015, Trejo appeared in a television commercial for Snickers that aired during Super Bowl XLIX, in which he portrayed Marcia Brady prior to eating the Snickers candy bar. In 2016 and 2017, he appeared as himself in transparent disguises in TV ads for Sling TV.
In 2017, Trejo played the role of 'Muerte' in Cross Wars and the 2019 film Cross: Rise of the Villains respectively. Trejo appeared on Hell's Kitchen twice: once as a guest diner in Season 16's final dinner service and once as a guest judge for the team challenge in the Season 21 episode "Everyone's Taco'ing About It". On August 6, 2017, Trejo made a guest appearance on season three of the Rick and Morty animated TV show, on the episode "Pickle Rick", in which he voiced the part of Mr. Jaguar. Together with Sasha Grey, he was a lead actor in Snapshot (2017), directed by Frankie Latina. That same year, he also appeared in Brooklyn Nine-Nine as Detective Rosa Diaz's father in an episode centered around Diaz's struggle to come out to her family.
In the TV show The Flash, he appeared in the episode Elongated Journey Into Night as the father of Cisco's love interest, Gypsy. His character works as a breacher (an interdimensional bounty hunter) who can manipulate the space-time fabric and travel to parallel worlds. Since 2018 he has voiced the role of Vasquez the bodyguard on the Disney show Big City Greens. In 2019, Trejo played the roles of Jose in Wish Man, Eduardo Hernandez in Grand-Daddy Day Care, Miguel in The Short History of the Long Road, Carlos in The Outsider, himself in Madness in the Method, Grave-digger in Bullets of Justice, Rondo in 3 from Hell and himself in Slayer: The Repent less Killogy. Also in 2019, Trejo had a supporting role in the film Acceleration, as Santos. Trejo also voiced the role of Clint Beltran in the Family Guy episode "Shanksgiving".