Studies on fish assemblages in streams point out, that understanding the relationship between species and their environment is crucial for conservation. The present study aimed at evaluating the effects of changes in abiotic variables on the composition and structure of fish assemblages in Neotropical urban streams from southern Brazil with different levels of urbanization. The composition and structure of fish assemblages showed significant differences along the urbanization gradient observed in the streams. Tolerant and non-native species were found in more urbanized sites. A matrix correlation revealed a relationship between abiotic variables and the spatial pattern of structure and composition of fish assemblages. Abiotic variables, such as total phosphorus, dissolved oxygen, and conductivity, determined the distribution of fish assemblages. Streams without urban influence exhibited intrinsically low species richness, suggesting that they are highly susceptible to species loss and diversity reduction. Thus, changes in water quality or hydrological conditions induced by urbanization may intensify these impacts.