In the documentary Death Diploma, Toole claimed he was forced to have sex with a friend of his father's when he was five years old. He felt he knew he was gay when he was 10, and claimed to have had a sexual relationship with a neighborhood boy when he was 12. Toole dropped out of school in the ninth grade and began visiting gay bars. He also claimed to have been a male prostitute as a teenager, and became obsessed with gay pornography. Toole claimed to have committed his first murder at the age of 14, when after being propositioned for sex by a traveling salesman, Toole ran over the salesman with his own car. Toole was first arrested at the age of 17 in August 1964 for loitering.

Much information on Toole between 1966 and 1973 is unclear, but it is believed that he began drifting around the Southwestern United States and that he supported himself by prostitution and panhandling. While living in Nebraska, Toole was one of the prime suspects in the 1974 murder of 24-year-old Patricia Webb. Shortly after, he left Nebraska and briefly settled in Boulder, Colorado. One month later, he became a prime suspect in the homicide of 31-year-old Ellen Holman, who was murdered on October 14, 1974. With many accusations against him, Toole left Boulder and headed back to Jacksonville.

In 1976, Toole met Henry Lee Lucas at a Jacksonville soup kitchen, and they soon developed a sexual relationship. Toole later claimed to have accompanied Lucas in 108 murders, sometimes at the behest of a cult called "The Hands of Death". Police, however, discounted the uncorroborated claim of the cult's existence.

In 1984, Toole confessed to two unsolved North West Florida slayings, including one of the I-10 murders. During an interview, he admitted to killing 18-year-old David Schallart, a hitchhiker he picked up east of Pensacola. Schallart's body, bearing five gunshot wounds in the left side of the head, was found on February 6, 1980, approximately 125 feet (38 m) off I-10's eastbound lane, 5 miles (8.0 km) east of Chipley. The second confession involved the death of 20-year-old Ada Johnson. Toole confessed that he shot her in the head on a road outside of Fort Walton Beach after kidnapping her at gunpoint at a Tallahassee nightclub. Psychiatrists Dr. Urbina and Dr. Sanches testified at Toole's 1984 Florida Supreme Court appeal that he was extremely impulsive and exhibited antisocial behavior as a result of a personality disorder and that he was a pyromaniac. The court found sufficient evidence that Toole was diagnosed with antisocial personality disorder.

Twenty-seven years after the 1981 murder of Adam Walsh, authorities officially named Toole as the likely killer.

On January 4, 1982, Toole barricaded 64-year-old George Sonnenberg in a boarding house where he was living in Jacksonville and set the house on fire. Sonnenberg died a week later of injuries he sustained in the fire. In April 1983, Toole was arrested for an unrelated arson incident in Jacksonville. For that crime, he was sentenced to 20 years in prison. While in custody, Toole confessed to killing George Sonnenberg. Toole signed a confession stating that he and Sonnenberg had begun a sexual relationship and, after the two had an argument, Toole lit Sonnenberg's home on fire.

On October 21, 1983, while imprisoned for two unrelated murders, Toole confessed to the 1981 murder of 6-year-old Adam Walsh. A few weeks after Toole made the confession, however, police investigating the case announced that they had lost Toole's impounded car and machete. John Walsh, Adam's father, continued to maintain that he believed Toole to be guilty. On December 16, 2008, Hollywood, Florida, police announced Toole as the murderer, and the Adam Walsh case was closed. The police did not reveal any new physical evidence and pointed out that they still had no DNA evidence.

During Toole's trial for murdering George Sonnenberg, Toole claimed that he did not light the home on fire and only signed the confession so he would be extradited back to Jacksonville. On April 28, 1984, a jury found Toole guilty of first degree murder and sentenced him to death. Later that year, Toole was found guilty of the February 1983 strangulation murder of a 19-year-old Tallahassee, Florida woman, and received a second death sentence; on appeal, however, both sentences were later commuted to life in prison.

The book Frustrated Witness, written by former Miami Herald Writer Willis Morgan, examines the Walsh case and cites circumstantial evidence that suggests Jeffrey Dahmer may have killed Adam Walsh. At the time of Walsh's murder, Dahmer was living a short drive away in Miami Beach and working at a sub shop where he had access to a blue van similar to the one seen leaving the mall after Walsh's disappearance. A number of witnesses reported seeing a man looking like Dahmer at the mall talking to young boys. When interviewed about Adam in the early 1990s, Dahmer repeatedly denied involvement in the crime, even stating; "I've told you everything—how I killed them, how I cooked them, who I ate. Why wouldn't I tell you if I did someone else?" After this rumor surfaced, John Walsh stated that he had "seen no evidence" linking his son's kidnapping and murder to Dahmer.

After his incarceration, Toole pleaded guilty to four more Jacksonville murders in 1991 and received four more life sentences.

On September 15, 1996, at the age of 49, Ottis Toole died at Florida State Prison of cirrhosis. As his body went unclaimed, he was buried in the Florida State Prison Cemetery.