Estuarine fish assemblages are often sensitive to environmental conditions, because fluctuation in physico-chemical conditions at different spatial and seasonal scales can directly influence species distributions. In this way, we conducted a field survey to investigate the role of estuarine gradient (environmental heterogeneity) in fish α and β diversity. The study was carried out in three zones in Mamanguape River estuary according to salinity and geomorphology features during an atypical climatic event in 2015. In total, 18,084 specimens of 125 species were captured. Additive partitioning of diversity analysis detected a higher proportion of beta diversity among estuarine zones during the rainy (β3 = 58.6%) and dry season (β3 = 40.94%) and were higher than expected by chance (Propexp> obs <0.001). Decomposing β-diversity analysis showed that total β-diversity (βsor) results were more dominated by species turnover (βsim) than nestedness (βnes) in both seasons. Forward selection procedure and db-RDA identified salinity, coarse sand and chlorophyll-a as the main environmental variables influencing βsor and site distance from estuary mouth and split as the main landscape variables. Variation partitioning analysis revealed more contribution to the pure fraction of environmental variables to fish species turnover, however, both pure fraction of environmental and landscape variables significantly contributed to βsim. Our study highlighted the importance to environmental heterogeneity and connectivity to promote fish diversity across the Mamanguape River estuary. Thus, future conservation policies should focus on maintaining these two components to guarantee its nursery ground role to estuarine fish assemblages.