This study aimed to determine the effect of the protein and antioxidant contents of edible mushrooms on the longevity of the fruit fly (Anastrepha ludens). The contents of protein (Bradford assay), antioxidants (DPPH and ABTS assays), total phenols, and flavonoids in nine strains of different edible mushroom species were determined. Freeze-dried and finely ground complete mushroom fruiting bodies were used to feed the flies, with a concentration of 0.5% in the diet. Male and female fruit flies, both fertile and sterile, were used in this study. Two controls were used: the standard fly diet and a diet supplemented with cinnamon as a food rich in antioxidants. Differences in protein and antioxidant contents were found among the evaluated strains. Differences were also observed in the responses of female and male flies as well as between the responses of fertile and sterile flies. Overall, the sterile flies lived longer. The addition of mushrooms in the diet resulted in greater longevity than in the controls. The use of sterile flies allowed observation of the effect of proteins and antioxidants on reproduction and the subsequent effect of reproduction on longevity.