Non-native species cause several impacts on freshwater biodiversity, but studies focusing on the Neotropical stream’s biota are still incipient. We used a data set of 586 headwater stream’s fish assemblages from the Brazilian Upper Paraná ecoregion to test whether the presence/absence of non-native species affect: species richness (S), functional diversity (MPD) and taxonomic diversity (Δ+). We compared diversity patterns of fish assemblages formed only by native species against those of assemblages formed by native and non-native species (Scenario 1); then, we removed non-native species from their original assemblages and recalculated their diversity values to compare them with those of fish assemblages formed only by native species again (Scenario 2). We also investigated: (1) whether non-native’s fish assemblages are associated with land use, topographic and watercourse connectivity variables; (2) fish ecological traits-environment relationship. In Scenario 1, S was higher in assemblages with the presence of non-native species, while in Scenario 2, both S and MPD were higher in assemblages where non-native species were removed. Non-native species were not directly related to land use, topographic or connectivity variables and most of them had a similar response to the environment when compared with native species. Findings show that non-native fish species are related to high-rich assemblages in headwaters, and they increase species richness and the functional redundancy of assemblages, decreasing functional diversity. Moreover, in most cases, native and non-native species seem to respond similarly to the environmental influence on their occurrence.