Abstract Our study assessed the fishers’ perception (local ecological knowledge, LEK) concerning environmental impacts on fisheries and fish species in the final portion of Sub-Middle and Lower São Francisco. The river was divided into four areas where 107 fishers from 22 locations (4 states) were surveyed. The dam was the most significant impact on fisheries. The loss of connectivity, changing the environmental complexity, and its secondary effects (e.g., flow control, interruption of migratory routes) were determining factors. Other impacts were pointed out, such as pollution, macrophytes, overfishing, non-native species, and aquaculture. Among migratory species, Pseudoplatystoma corruscans and Salminus franciscanus have been absent from fishery catches for decades. Prochilodus argenteus records in fisheries are likely associated with migration routes to small tributaries. The environmental disturbance favored the establishment of non-native species such as Oreochromis niloticus, Cichla monoculus, and Metynnis lippincottianus along the studied section. Over the final portion of the São Francisco River, the more significant seawater intrusion changed the target species for fisheries activities. Thus, our data validate the importance of fishers’ LEK and reinforce the adverse effects of the reservoir cascade on fishing and migratory fishes.