Fish are subject to diverse stimulus in floodplain rivers resulting from interactions between environmental factors. In such complex and interconnected ecosystems, fish assemblages are thus highly dynamic across space and time. Here, we analysed fish assemblage structures in lotic/lentic habitats of a geomorphic unit of the Middle Paraná River during summer, autumn, and winter of seven consecutive years. We observed that three main factors trigger significant structural changes: seasonality, habitat characteristics, and floodplain connectivity. The last one appears to have a determinant role on fish structures. Processes of connection/isolation of floodplain environments produced significant variations in fish abundance and species richness.Main results showed that some ecological processes can be largely explained by connectivity variations, such as fish dispersal in the floodplain, fish concentration in remnant floodplain lakes, and migrations back to permanent floodplain water bodies.This study provided a quantitative measurement of connectivity to understand fish dynamics in a giant floodplain and highlights the importance of similar investigations to preserve the fish fauna in these rather unique ecosystems.