Aquatic macrophytes produce large amounts of organic matter and have an essential structuring role in floodplains. This process highlights the importance of this community to aquatic biodiversity maintenance. We investigated the role of a flood disturbance on the response of macrophyte assemblages in regional and local structuring in the Upper Paraná River floodplain. Plant species were recorded before (November 2006) and after (March 2007) an uncommon increase in water level caused by the El Niño South Oscillation, which is considered a disturbance. Samples were taken in lakes and backwaters located in the floodplain and connected to three distinct rivers (that differentiate three sub-systems). Species richness and the assemblage structure of macrophyte patches underwent significant changes after the flood disturbance, depending on the specific sub-system (rivers) to which the lakes were connected. In addition, flood disturbance had a strong impact on community organization at the local scale. However, regionalization with respect to sub-systems remained significant after the flood disturbance. Our results emphasize the importance of connection to the river on macrophyte community composition and richness, and suggest that flood events in the Upper Paraná River floodplain disrupt community organization only at fine (local) scales.