The Neotropics represent a hotspot for freshwater biodiversity with vast number of fish species of scarce ecological knowledge. This holds true for the Uruguay River, where fish assemblages and their diets remain unexplored. Fish assemblages were surveyed in 14 sites along the river main course, from headwaters to mouth (approximately 1800 km), with the aim to identify the trophic roles of fishes and to describe trophic structure of these assemblages, following standardized sampling campaigns and laboratory procedures. One hundred species (2309 gut contents) were analysed and classified into four trophic groups subdivided into eight lower-level groups: Piscivore, piscivore-invertivore, detritivore, omnivore-detritivore, omnivore-invertivore, omnivore-planktivore and omnivore-herbivore. The trophic structure of the assemblages varied along the river, with the relative species richness of fish consuming terrestrial invertebrates increasing towards the middle river section, probably driven by the large floodplains in that areas, supporting global theories such as the flood pulse concept. This study describes the feeding habits of fish along the Uruguay River, being the first dietary description for 29 species. This knowledge is essential for management and conservation, serving as baseline in the context of future environmental changes while generating novel evidence on the functioning of ecosystems in this scarcely studied climatic region.