Resumo. Abstract Aim This study aimed to investigate the diet of seven species of fish that consume predominantly benthic macroinvertebrates, and the availability of these organisms in the environment. We analyzed the occurrence of trophic segregation between species and the correlation between the abundance of macroinvertebrates in the diet and in the environment. Methods Fish and macroinvertebrates were sampled in three streams of the Pirapó River basin (Upper Paraná River - Brazil). Differences in diet composition between species were tested using a multivariate permutation analysis of variance (PERMANOVA). A Spearman Correlation was performed to test the relation between the abundance the macroinvertebrates consumed and those available in the environment. Results All species mainly consumed aquatic insect, and significant interspecific variations in diet composition were found for most of them. For five out of the seven species of fish analyzed no significant correlation was detected between the abundance of macroinvertebrates in the environment and in the diet. Conclusions The results showed that most of the species segregate and their diets are not positively correlated with the abundance of preys in the environment, indicating that the most consumed macroinvertebrates were not the most abundant in the environment. These results suggest that the fish species select the macroinvertebrates, and that the morphological and behavioral characteristics of both predators and prey are important in feeding fish from streams.