Determining how assembly rules (e.g. limiting similarity, environmental filtering and neutrality) shape community structure along environmental gradients and across spatial scales is still controversial. The study of functional relationships between coexisting species may help to disentangle among these assembly rules. Here, we compared pairwise functional dissimilarities between fish species to their corresponding pairwise co-occurrences. Fish assemblages (n = 835) were sampled monthly in Patos-Mirim system (Brazil) using both bottom trawling and beach seining. Species occurrences were recorded and functional traits related to locomotion and food acquisition were measured on several individuals from each species. The region studied was divided in two areas corresponding to each side of a floodgate located in São Gonçalo Channel: a freshwater channel up the floodgate and, down the floodgate, the Patos Lagoon estuary. The relationship between functional dissimilarity and co-occurrence between species pairs was assessed using quantile regressions for each month and at different spatial scales. Overall, quantile regression coefficients between functional dissimilarities (either based on locomotion types or feeding habits) and co-occurrence values were negative, suggesting that co-occurrence increases with functional redundancy regardless of spatial scale. Our results support the assumption that environmental filters have more influence than biotic interactions on the structure of fish assemblages even locally.