Fish otoliths store geochemical and isotopic signatures that can be used as proxies for environmental conditions in ecological investigations. We investigated the δ13C and δ18O of otoliths of four freshwater fish species (Astronotus ocellatus, Serrasalmus brandtii, Plagioscion squamosissimus, and Cichla ocellaris) from the Moxotó Reservoir in the São Francisco River, Brazil. The enriched δ13C signatures that distinguish A. ocellatus from other species could be caused by dissolved inorganic carbon (δ13CDIC) in reservoir waters. Macrophytes growing along the reservoir margins would favor photosynthetic uptake of 12C, leading to enriched δ13CDIC incorporated into the otoliths of those fish. Otolith δ18O signatures appear to reflect water column preferences, in which species preferentially inhabiting surface waters (such as S. Brandtii) show more enriched values (due to high surface evaporation ratios), while bottom water species show depleted δ18O values. Our results represent the first investigation of the isotopic compositions of fish otoliths in freshwater environments in the São Francisco River, and shed light on the interpretation of isotopic information stored in otoliths and how they can be used to infer the ecological strategies of freshwater fish.