Impoundments alter connectivity, sediment transport and water discharge in rivers and floodplains, affecting recruitment, habitat and resource availability for fish including benthic invertivorous fish, which represent an important link between primary producers and higher trophic levels in tropical aquatic ecosystems. We investigated long-term changes to water regime, water quality, and invertivorous fish assemblages pre and post impoundment in three rivers downstream of Porto Primavera Reservoir in south Brazil: Paraná, Baía and Ivinhema rivers. Impacts were distinct in the Paraná River, which is fully obstructed by the dam, less evident in the Baía River which is partially obstructed by the dam, but absent in the unimpounded Ivinhema River. Changes in water regime were reflected mainly as changes in water-level fluctuation with little effect on timing. Water transparency increased in the Paraná River post impoundment but did not change in the Baía and Ivinhema rivers. Changes in fish assemblages included a decrease in benthic invertivorous fish in the Paraná River and a shift in invertivorous fish assemblage structure in the Baía and Paraná rivers but not in the unimpounded Ivinhema River. Changes in water regime and water transparency, caused by impoundment, directly or indirectly impacted invertivorous fish assemblages. Alterations of fish assemblages following environmental changes have consequences over the entire ecosystem, including a potential decrease in the diversity of mechanisms for energy flow. We suggest that keeping existing unimpounded tributaries free of dams, engineering artificial floods, and intensive management of fish habitat within the floodplain may preserve native fish assemblages and help maintain functionality and ecosystem services in highly impounded rivers.