This is a list of main and recurring fictional characters from The Venture Bros., a comic science fiction television series aired on Adult Swim from 2003 to 2018.

Team Venture consists of the central characters in the show; they previously resided in a fortified compound in Colorado Springs which also served as the headquarters for their company, "Venture Industries." Following the destruction of the compound in Season Six and Dr. Venture's inheritance of his brother's fortune, they relocate to the Ventech Tower at Columbus Circle in Manhattan.

Dr. Thaddeus "Rusty" Venture (voiced by James Urbaniak): Dean and Hank's father, and head of Venture Industries. He is a capable scientist and inventor, but his greed, lack of formal education, and insistence on cutting corners result in his attempts at technological innovation failing, causing chaos for him, his family, friends, and sometimes the world at large. Although the money he earns is sufficient to support him and his family, it fails to satisfy his greed and his aspirations to match his father's wealth and success. While he cares for his sons, his father's abusive and negligent parenting influences the way he treats them, sometimes showing as much indifference to them as his father did to him. In Season 7, it is revealed that Dr. Venture is not the original, but rather a clone of the first Rusty Venture, who died in the past.

Brock Fitzgerald Samson (voiced by Patrick Warburton): Born in Omaha, Nebraska, to a single mother, Brock is a hypermasculine one-half Swedish, one-quarter Polish, one-quarter Winnebago "murder machine" who serves as a bodyguard to the Venture family. He attended the same university as Dr. Venture until he was expelled for accidentally killing one of his football team's teammates. Despite holding a license to kill, Brock eschews guns and instead favors a Ka-Bar fighting knife as his signature weapon. He grapples with his role in the world and his purpose, guided by a personal code of ethics concerning innocents and whom he will eliminate if they pose a threat to himself or the Ventures. Brock owns an orange 1969 Dodge Hemi Charger named "Adrianne", frequently seen driving or shirtlessly washing at the Venture compound. Following the explosion of the car carrying Henchman 24, H.E.L.P.eR.'s head was briefly embedded in Brock's chest, leading to 24's death; the head was later removed and replaced with a metal plate.

Henry Allen "Hank" Venture (voiced by Christopher McCulloch): Hank, the elder fraternal twin with blond hair, is a teenage boy and one half of the titular duo. He emerges as the more extroverted and adventurous of the pair, drawn to action and thrill-seeking, often envisioning himself as a vigilante superhero. In season 4, he exhibits increased rebelliousness following Brock's departure, even losing his virginity to an older woman who happens to be his best friend's mother. After briefly affiliating with former Henchman 21's rendition of S.P.H.I.N.X., Hank acquires the power suit of former S.P.H.I.N.X. member The Countess, donning it until it is stolen and destroyed. This incident leads to Hank undergoing physical therapy due to muscle atrophy. In season 6, he opts out of college and takes on a job as a delivery boy at a pizzeria. Additionally, he embarks on a romantic relationship with Sirena Ong, the daughter of his father's current Guild-assigned villain, Wide Wale. In season 7, Hank slips into a coma after discovering Sirena's infidelity with Dean but eventually recovers.

Dean Venture (voiced by Michael Sinterniklaas): As a teenage boy and one half of the eponymous pair, Dean is the younger fraternal twin with red hair. Despite being more timid than Hank, he exhibits signs of greater emotional maturity. Dean garners more favor from their father, who grooms him to succeed as the heir to Venture Industries. Like his brother, Dean has faced death over a dozen times, only to be replaced by an exact clone each time, devoid of memories of his demise. Following a breakup with Triana and the revelation of his clone status, Dean adopts a more emo persona, donning a black "speedsuit" and relocating from his childhood bedroom, displaying reluctance to join family adventures. However, Hank's optimism and familial support pull him out of his emotional slump. In season 6, Dean enrolls at the fictional Stuyvestant University in New York, excelling as a philosophy student and befriending Brown Widow, his roommate. In season 7, he engages in an affair with Hank's girlfriend, Sirena Ong, but later regrets his actions.

H.E.L.P.eR. (voiced by Christopher McCulloch; credited as "Soul-Bot"): H.E.L.P.eR., short for Humanoid Electronic Lab Partner Robot, serves as the Venture family's helper robot, communicating through electronic beeps. Despite his sensitivity, the Ventures often regard him as an old appliance or pet. Throughout their adventures, H.E.L.P.eR. frequently gets smashed to pieces but miraculously appears repaired by the next episode.

Sergeant Hatred, also known as Courtney Robert Haine, is voiced by Brendon Small in season 2 and Christopher McCulloch from season 3 onward. He serves as Brock's former supervisor in the O.S.I., the husband of Native American villainess Princess Tinyfeet, and the Venture family's bodyguard. He is a large, muscular man with a giant red 'H' tattooed on his face, often seen wearing military fatigues. Despite being a convicted sex offender for having slept with a 17-year-old, he is liked and respected throughout the supervillain community for his unfailing politeness and generosity; certain episodes also reveal that he knows his sexual attraction to teenagers is wrong, and tries to keep it in check using medication and electroshock therapy. He is known for his caring nature beneath his battle-hardened exterior.