By 1920, Dorothy Mackaill had begun making the transition from “Follies Girl” to film actress. That same year she appeared in her first film, the Wilfred Noy-directed mystery, The Face at the Window. Mackaill also appeared in several comedies of 1920 opposite actor Johnny Hines. In 1921 she appeared opposite Anna May Wong, Noah Beery, and Lon Chaney in the Marshall Neilan-directed drama Bits of Life. In the following years, Mackaill would appear opposite such popular actors as Richard Barthelmess, Rod La Rocque, Colleen Moore, John Barrymore, George O’Brien, Bebe Daniels, Milton Sills and Anna Q. Nilsson. In 1924, Mackaill rose to leading lady status in the drama The Man Who Came Back, opposite rugged matinee idol George O’Brien. Her role of the nightclub chanteuse Marcelle catapulted Mackaill into a genuine Hollywood star and her career continued to flourish throughout the remainder of the 1920s. In early 1924 she starred in the western film, The Mine with the Iron Door, shot on location outside of Tucson, Arizona. That same year she was awarded the WAMPAS Baby Stars award by the Western Association of Motion Picture Advertisers in the United States, which honored thirteen young women each year who they believed to be on the threshold of movie stardom. Other notable recipients of the award that year were Clara Bow, Julanne Johnston and Lucille Ricksen.

Dorothy Mackaill made a smooth transition to sound with the part-talkie The Barker (1928) and had success in talkies for the next couple of years. First National Pictures was acquired by Warner Brothers in September 1928, and her contract with First National was not renewed upon its expiration in 1931. Her most memorable role of this era was the 1932 Columbia Pictures release Love Affair with a young Humphrey Bogart as her leading man. She made several films for MGM, Paramount and Columbia before retiring in 1937 to care for her aging mother. In 1955, Mackaill moved to Honolulu where she remained for the rest of her life. She had fallen in love with the islands while filming His Captive Woman in 1929.  Mackaill lived at the luxurious Royal Hawaiian Hotel on the beach at Waikiki as a sort of celebrity in residence and enjoyed swimming in the ocean nearly every day.  She occasionally came out of retirement to appear in roles for television, notably in two episodes of Hawaii Five-O in 1976 and 1980, which was filmed on location in Hawaii. Dorothy Mackaill died of kidney failure in Honolulu, Hawaii on August 12, 1990, aged 87. She was cremated and her ashes were scattered at sea off of Waikiki Beach.