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Summary Major hydroelectric dams are proliferating in tropical regions such as Amazonia, where extensive new hydropower developments are planned despite potentially severe ecological and social impacts. The status of freshwater biota in the vicinity of existing dams could be valuable to predict the effects of such developments, but detailed ecological monitoring before and after dam construction is frequently lacking. In the absence of these data, we used a space‐for‐time replacement to compare a key component of the aquatic biota at upstream and downstream sites, with the latter more closely resembling river channel conditions prior to the dam. We assessed the fish assemblages upstream and downstream of the Coaracy Nunes Dam in Amapá, Brazil, the first ecological study at this site since this dam was constructed 42 years earlier. We used gillnets during eight bimonthly field campaigns, covering both wet and dry seasons, and sampled the ichthyofauna (1819 individuals, 81 species) at four sites: Downstream Channel, Reservoir, Upstream Lake and Upstream Channel. We found clear negative impacts on the abundance, biomass, species richness, alpha diversity and species dominance upstream of the dam. The physical subdivision of the river channel and the upstream channel conversion from lentic to lotic habitats were strongly associated with differences in the composition and structure of fish communities. Notable changes include an increased contribution of small‐bodied fish in the reservoir and an absence of long‐distance migrants upstream of the dam. Downstream channel habitats, in particular, retained their fish diversity with high conservation value typical of eastern Amazonia, yet these areas now face the threat of new hydropower development farther downstream. The long‐term impacts on aquatic biodiversity highlighted in this study are especially relevant in the face of burgeoning new hydroelectricity development plans for rivers across lowland Amazonia.