Resumo. Abstract Aim We investigated the long-term effects of a rural landscape on the structure and trophic organization of a fish assemblage. Methods We compared environmental data and data from fish assemblage structure and trophic organization sampled in a stream located in a rural area, within a time gap of 20 years. Results We observed only punctual changes in the environmental variables and fish structure, which may not be related to the rural landscape. In fish diet, insectivory remained predominant in all sample periods. However, when we analyzed the groups of hexapods consumed by the fish species, we found a substitution of Ephemeroptera and Trichoptera by Diptera over time. Conclusions Although the fish assemblage structure was not affected, the insectivore diet analysis showed that the stream stretch could be in an ongoing process of environmental quality loss, once the simplification in the macroinvertebrates assemblage structure over time is indicative of environmental degradation.