Non‐native species represent a significant threat to biodiversity, particularly in aquatic environments where they are often introduced for purposes such as fishing or the aquarium trade. This study aimed to evaluate the influence of isolation and the size of federal protected areas (PAs) on the number of non‐native and native freshwater fish species in Brazilian PAs. Data from Brazilian PAs were gathered, including year of creation, buffer of human impacts around the PA, PA categories, coverage by freshwater, tourist visits, sampling sites, geographical coordinates (predictor variables) and information on the number of non‐native and native freshwater fish species (response variables). Linear regression analysis was employed to examine the influence of predictor variables on the number of non‐native and native fish species. A total of 33 different non‐native fish species were identified, with Poecilia reticulata, Hyphessobrycon eques, Hoplias lacerda and Cichla kelberi being the most widespread, occurring in 15, 7, 5 and 4 distinct PAs, respectively. The overall fish biodiversity within PAs encompassed 2670 species, with the National Park of Anavilhanas and the Environmental Protection Area of Ilha e Várzea do Paraná exhibiting the highest species richness. Greater human impact, larger coverage by freshwater and increased sampling effort were positively correlated with the number of recorded non‐native species. Factors such as year of creation, type of PA and tourist visitation did not have a significant effect. In contrast, native species exhibited a higher number of occurrences in PAs with lower human impact, longer histories, larger freshwater coverage areas and more extensive sampling efforts. These findings underscore the necessity for comprehensive management strategies to mitigate the impact of non‐native species on biodiversity within PAs.