McAfee was born in the Forest of Dean, Gloucestershire, United Kingdom on September 18, 1945 on a U.S. Army base to an American father, who was stationed there, a British mother, and raised in Salem, Virginia. He received a bachelor's degree in mathematics in 1967 from Roanoke College, which subsequently awarded him an honorary Sc.D. degree in 2008.

McAfee was employed as a programmer by NASA's Institute for Space Studies in New York City from 1968 to 1970. From there he went to Univac as a software designer and later to Xerox as an operating system Architect. In 1978 he joined Computer Sciences Corporation as a software consultant. He worked for consulting firm Booz Allen Hamilton from 1980–1982. In the 1980s, while employed by Lockheed, McAfee received a copy of the Brain computer virus and began developing software to combat viruses.

In 1987 McAfee founded McAfee Associates, a computer anti-virus company. In 1989, he quit Lockheed and began working full-time at McAfee Associates, which he initially operated from his home in Santa Clara, California. The company was incorporated in Delaware in 1992, and McAfee resigned from the company in 1994. Two years after McAfee Associates went public, McAfee sold his remaining stake in the company.

Network Associates was formed in 1997 as a merger of McAfee Associates and Network General. The Network Associates company name was retained for seven years, when it was renamed McAfee, Inc. In August 2010, Intel bought McAfee, maintaining the separate branding, until January 2014, when it announced that McAfee related products will be marketed as Intel Security. McAfee expressed his pleasure at his name no longer being associated with the software.

Other Business ventures that were founded by McAfee include Tribal Voice, which developed one of the first instant messaging programs, PowWow. In 2000, John McAfee invested in and joined the board of Directors of Zone Labs, makers of firewall software, prior to its acquisition by Check Point Software in 2003.

In 2009, McAfee was interviewed in Belize for the CNBC special "The Bubble Decade", in which it was reported that he had invested in and/or built many mansions in the USA that went unsold when the 2007 global recession hit. The report also discussed his quest to produce plants for possible medicinal uses on his land in Belize.

In February 2010, McAfee started the company QuorumEx, headquartered in Belize, which aimed to produce commercial all natural antibiotics based on anti-quorum sensing Technology.

On December 6, 2012, Reuters and ABC News reported that McAfee had two minor heart attacks in a Guatemalan detention center and was hospitalized. McAfee's Lawyer stated that his client had not suffered heart attacks, but had instead suffered from high blood pressure and anxiety attacks. McAfee later said that he faked the heart attacks while being held in Guatemala to buy time for his attorney to file a series of appeals that ultimately prevented his deportation to Belize, hastening the government's decision to send him back to the United States. On December 12, 2012, McAfee was released from detention in Guatemala and deported to the United States.

In early 2013, McAfee was residing in Portland, Oregon, but he has since moved to Lexington, Tennessee, with his wife Janice Dyson.

As of January 2014, Belizean police have presented no new accusations, and they have not persisted in seeking McAfee's imprisonment for any of the crimes of which they accused him. However, they have auctioned off McAfee's seized assets, and his home was burned down under suspicious circumstances.

On August 2, 2015, McAfee was arrested in Henderson County, Tennessee, on charges of one count of driving under the influence and one count of possession of a firearm while intoxicated.

Gringo: The Dangerous Life of John McAfee is a Showtime Networks documentary about the portion of McAfee's life spent in Belize. It began airing in September 2016. The documentary contains allegations of both the sexual assault of McAfee's former Business partner, Allison Adonizio, and the murder of his neighbour, American expat Gregory Faull. In an interview with Bloomberg's Pimm Fox and Kathleen Hayes on September 8, 2016, McAfee claimed that these incidents were fabricated, saying that "Belize is a third-world Banana republic and you can go down there and make any story you want if you pay your interviewees, which Showtime did."

On May 12, 2017, McAfee was interviewed in ABC's 20/20 regarding the alleged murder of his neighbor, Greg Faull. During the hour, Janice McAfee, John's wife, was also interviewed.