The museum is dedicated to the contentious relationship between organized crime and law enforcement within the historical context of Las Vegas and the entire United States.

[6] The idea faced early opposition from Italian-American groups, while being supported by the FBI, including the former head agent in Las Vegas, Ellen Knowlton, who joined as president of the museum's board.

[8] The centerpiece of the Mob Museum is the second floor courtroom, which was the location of one of fourteen national Kefauver Committee hearings to expose organized crime held in 1950 and 1951.

The museum offers a wide array of thematically oriented sections addressing the Mob's involvement in illegal activities such as gambling, drugs, prostitution or bootlegging and the efforts of law enforcement to counteract and eliminate those criminal operations.

Additionally the visitor is able to view some uniquely disturbing, otherwise rarely accessible material, for example the photographs of victims of the most famous murders credited to the Mafia (appropriately named "Mob's Greatest Hits"; these are quite graphic in nature, because they show the actual corpses of the deceased), as well as pictures and short biographies of the most popular and notorious gangster personalities.

Using photos, text, displays, interactive techniques, hands-on exhibits, and other first-class museum methods, the visitor learns about the history of organized crime, Prohibition and the business opportunity it provided.

Visitors learn about Las Vegas' first casinos, Howard Hughes, J. Edgar Hoover, the origins of the FBI, Al Capone, Eliot Ness, and much more.