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With the aim of assessing whether hydrological alterations differently affect non-native and native fish species in coastal systems, we investigated fish assemblages along the fluvio-estuarine ecotone of the Neotropical São João River, Brazil. During a year, samplings with standardized efforts and limnological parameter records were carried out bimonthly at 15 sampling points in the last 50 km of the São João River, representing natural (meandering riverine and estuarine) and modified (reservoir, channelized and drained floodplain) stretches. We recorded 48 marine and 51 freshwater fish species, with 12 non-native species among the latter. Several native species were exclusively found within natural stretches, whereas non-native species presented significantly higher biomass catches in modified stretches. Only three species occurred in the entire study area, all of which were non-native species. Non-native species were associated with low dissolved oxygen and pH levels below the dam, whereas native freshwater species responded negatively to water transparency, and marine species responded positively to water conductivity at the hydrologically unaltered points. The hydrological alterations disrupted the fluvio-estuarine ecotone of the lower São João River, which plays the role of a dispersion source for non-native species and where invasive species are favored. [ABSTRACT FROM AUTHOR]