Giant otters Pteronura brasiliensis are semiaquatic mammals that mainly eat fish, the abundance of which is affected by seasonal flooding and habitat structure. The piscivorous habits of giant otters lead to negative human perception and conflicts with fisheries. We compared giant otter feeding habits between seasons and habitats in the Southern Pantanal, Brazil, by analyzing feces collected between September 2008 and June 2011.We investigated whether habitat and season affected P. brasiliensis diet composition and prey size. We calculated the frequency of occurrence, relative frequency, and overlap of fish species eaten by giant otters and caught by fishermen. The giant otters had a more diverse assemblage of fish prey than the offtake in the fisheries. We did not find strong seasonality in otter diets, but diet composition and prey size differed between rivers and lakes. The giant otter diet had higher overlap with the offtake of sport than with professional fishermen. Although the otters' piscivorous diet often leads to negative perceptions by humans, the low overlap between otter diet and species taken in local fisheries suggests that otters have little effect on the commercial fishery. These results indicate that educational programs could be used to reduce perceived conflict between giant otters and fishermen.