One of the main aspects of natural history is trophic ecology, which affects survival and population size. In the present study, we registered the contribution of prey types to the diet of Physalaemus ephippifer and tested for differences in diet according to sex and season (rainy and dry) of three populations of P. ephippifer in eastern Amazonia. We analyzed frequency of occurrence, trophic amplitude, feeding overlap, and food importance index for each prey category. We analyzed 102 specimens (69 males and 33 females) and observed that the most important prey categories were termites and ants. The most important prey category for females during rainy season included coleopteran larvae, whereas during dry season the most important prey category was termites. In both seasons, the most important prey category for males was ants. We did not observe any seasonal or sexual variation in the diet of P. ephippifer. On the other hand, during the rainy season, coleopteran larvae and termites were the most important prey categories for females, which have a more nutritious diet than males. Increase in nutritional value of the diet of females coincided with the reproductive period, when females would require a greater amount of energy for gamete production.