Wilson appeared in over twenty motion pictures, but won immortality for his role as Sam in the 1942 film . For his role, he was paid $350 a week for seven weeks. Sydney Greenstreet, in comparison, was paid $3750 a week.

Sam is a singer and pianist employed by nightclub owner Rick (Humphrey Bogart). The Herman Hupfield song "As Time Goes By" appears as a continuing musical and emotional motif throughout the film. Rick and Ilsa (Ingrid Bergman) regard it as "their song" and associate it with the days of their love affair in Paris. Because of their breakup and Ilsa's marriage to another, Rick has forbidden the song to be played in his club. When Ilsa appears in his nightclub she requests it and Sam acquiesces. Dooley Wilson gives a genial and warm rendition of the song. The performance is remembered for itself, as well as for its cinematic associations. The song makes Rick aware of Ilsa's presence and her continuing feelings for him. According to Aljean Harmetz, said he created "something joyous."

In a later scene, Rick sits in a darkened nightclub, alone except for Sam, drinking heavily and torturing himself by insisting that Sam repeatedly play the song, saying "You played it for her, you can play it for me... If she can stand it, I can! Play it!" This is the line often misquoted as "Play it again, Sam!"

In the film, Wilson as Sam performs several other songs for the cafe audience: "It Had To Be You", "Shine", and "Knock On Wood".

Wilson was a singer and drummer, but not a pianist. Sam's piano playing in the film was actually performed by Elliot Carpenter, who was placed where Wilson could see and imitate his hand movements. The only black people on the