Born in New York's "Little Italy," Prestopino followed the educational path typical of many immigrant children: elementary school and then a few years at a vocational high school. After having pursued such a path, in which he attempted to learn sign painting, he left school at the age of fourteen. At another fixture of immigrant life, the settlement house, he was introduced to the fine arts, winning a scholarship to the National Academy of Design. There he was a student of Charles Hawthorne and became acquainted with the work of the Ashcan School painters.
After his studies he established a studio in New York and began painting the life he saw around him. A series of stays at the MacDowell Colony in Peterborough, New Hampshire, beginning in the summer of 1934, had a marked influence on his work. He spent eight summers in residence there, during one of which he met his wife, Elizabeth. Along with depictions of working-class city dwellers, he now began painting scenes that embodied "small town individualism and egalitarianism." During the 1960s and '70s, Prestopino actively supported the civil rights and antiwar movements, donating the sales of his art and designs to benefit these causes. His later work moved away from the figure and toward abstraction.
William H. Truettner and Roger B. Stein, editors, with contributions by Dona Brown, Thomas Andrew Denenberg, Judith K. Maxwell, Stephen Nissenbaum, Bruce Robertson, Roger B. Stein, and William H. Truettner Picturing Old New England: Image and Memory (Washington, D.C.; New Haven, Conn; and London: National Museum of American Art with Yale University Press, 1999)