Resumo. Abstract Aim This study investigated changes in the dietary composition and trophic niche breadth of Eigenmannia desantanai in the context of hydrological periods, rivers, and ontogeny. Methods Collections were performed monthly on the Paraguay and Amonguijá Rivers between February 2009 and January 2011. Results A total of 338 specimens of E. desantanai were collected, 224 of which came from the Amonguijá River and 114 from the Paraguay River. In all, 21 food items were identified. Fully remains of digested animals (RDA), Chironomidae, Cladocera, and Ostracoda showed greater importance in the diet of E. desantanai. Dietary composition was influenced by hydrological periods, followed by differences between sampled rivers, and ontogeny. In contrast, only hydrological periods explained variation in the E. desantanai trophic niche breadth. Conclusions The diet of E. desantanai is mainly composed of Chironomidae, Cladocera, and Ostracoda; however, individuals consumed other food items owing to changes in hydrological periods, followed by characteristics of the Amonguijá and Paraguay Rivers and ontogenetic variation. In this respect, the results of this study are germane to the food ecology of E. desantanai, providing guidelines for the management and conservation of the species.