Freshwater ecosystems are facing mounting challenges. The widespread introduction of non-native species, for example, has resulted in the loss of native species and the substantial reconfiguration of diversity patterns across regions. Documenting such impacts remains critical for informing national-level biosecurity policies. Here, we explore changes in biogeographic patterns in freshwater fish diversity in response to the spread of non-native species, teasing apart the geographic (watersheds) and taxonomic (species) drivers of patterns at the global scale. We leveraged global databases of fish species occurrence to estimate the unique contributions of local watersheds and species (native and non-native origin) to beta-diversity for biogeographic domains. Beta-diversity metrics of watersheds and species at a domain scale can be interpreted as their importance for the uniqueness in freshwater fish composition. We report significant changes in freshwater fish beta-diversity in response to non-native species, with the largest impacts in the Ethiopian, Nearctic and Palearctic domains, even though non-natives decreased the contribution of watersheds to beta-diversity in all domains, particularly in watersheds with known impacts. Watersheds identified as most important for promoting beta-diversity were not evenly distributed across domains, were influenced by geographical isolation and their unique compositions were composed of many endemic and threatened species. Highest values of species contributions to enhancing beta-diversity were mainly observed for native and threatened species, although mean values of species contributions were higher for non-threatened species. Species from the most important watersheds had wide ecological tolerances, were, in general, natives, endemics and/or with IUCN threat status. Our findings underscore the widespread consequences of non-native species for shaping biogeographic patterns of freshwater fishes in the Anthropocene.