Interest has grown in how birds respond to environmental characteristics of river systems, motivated by the high biodiversity and biomass of waterbirds in these systems and the importance of waterbirds in hunting for sport or sustenance. We studied the influence of natural (water level fluctuations and environmental heterogeneity) and human-related (cattle grazing and hunting) factors on waterbirds in the Pre-Delta region of the Paraná River system during three years. Our objectives were to evaluate which are the main environmental factors that influence the assemblages of waterbirds and whether these variables explain differences in waterbird assemblages between protected and non-protected areas. Both species richness and abundance of waterbirds increased with the surface of open water and the number of environmental units. The larger surface of open water in the non-protected area contributed to explaining the higher species richness and abundance of waterbirds, including of species with hunting pressure, in this area. During flood stages the protected area maintained a high abundance of water birds, probably because the higher proportion of trees and aquatic vegetation in this area provided perches and foraging sites. We recommend enlarging protected areas in the Pre-Delta region because of their role of sheltering waterbirds during floods. Our results showed that the presence of both protected and non-protected areas increased the diversity of waterbirds in the region due to the species turnover, so it is recommendable to keep a mixture of both in the region.