The study was done in a first order stream in the southern portion of the Brazilian Atlantic Rain Forest. Samples of the aquatic megafauna (amphibians, crustaceans and fishes) were taken with the aim of describing spatial (longitudinal) and temporal (seasonal) patterns in species composition and abundance. Thirty four structural and limnological variables at macro and mesoscales from three sampling reaches were analysed. The spatio-temporal analysis of species richness and diversity indicated a gradient in which values increased in an upstream–downstream direction, independently of the season of the year. The results showed a strong influence of structural environmental variables on community structure. Furthermore, they revealed a hierarchical relation between macroscale and mesoscale variables and their influence on community abundance and composition in the various spatio-temporal sampling units analysed. The spatial distribution of species richness and diversity in the Carvo creek was strongly influenced by the presence of waterfalls, being progressively richer and more diverse downstream. Waterfalls seem to function as selective filters more than as absolute barriers, presenting different efficiencies for different species.