The flood pulse regime and the hydrological connectivity determine the lateral bidirectional exchanges of water, chemical compounds, and biota between the river and the floodplain habitats. The primary goal of the present research was to analyze the effect of water flow on macroinvertebrates in two water levels in a lateral connectivity gradient, from the main channel through a connection channel to a permanently connected lake. We tested the hypothesis that the water flow from the main channel to the floodplain habitats during high water level causes a decrease in beta diversity between the sites, increasing similarity in the system. To test this hypothesis, we sampled a river–floodplain–lake system of the Middle Paraná River during two water levels, and analyzed the spatial and temporal turnover of species between sites and habitats. Local physical characteristics, such as depth, benthic particulate organic matter, and grain size of bottom sediments influenced assemblage composition. Taxa richness, density, and Shannon diversity differed among habitats within the river–lake system, but did not show significant differences between water levels. Richness, density, and diversity were higher in the lake and the connection channel than in the Paraná River bank. Beta diversity was significantly higher during high water period. During low water period, benthic assemblage composition was homogenized, as reflected by the lower values of species turnover between the sites situated in the main channel–lake corridor during this phase. The lateral bidirectional exchanges among the habitats are essential for maintaining the specific invertebrate diversity of large river corridors.