Although diazotrophs play a critical role in the nitrogen (N) cycle by supplying new N, the response and mechanism of nifH gene diversity and community structure to minor changes in salinity remain unclear. Here, we investigated the diazotrophic communities in both rhizospheric and non-rhizospheric soils along a low-level salinity gradient across one freshwater (0.08 ± 0.02‰) and two oligohaline (1.27 ± 0.09 and 3.31 ± 0.14‰) wetlands by sequencing the nifH gene. The nifH gene alpha diversity was similar along the salinity gradient, but community structure differed markedly. The most abundant genera across the samples were within the Deltaproteobacteria class, including Geobacter, Desulfuromonas, Pelobacter, and Desulfovibrio. The nifH gene diversity (both alpha and beta diversity) and community structure did not differ significantly between rhizospheric and non-rhizospheric soils. Furthermore, soil salinity, pH, and nutrient availability (i.e., NH4+-N, NO3−-N, and C:N ratio) had synergistic effects on the diazotrophic community structure. In conclusion, the transition from freshwater to oligohaline water significantly altered the diazotrophic community structures but not diazotrophic diversity. Our study shows that saltwater intrusion may affect estuarine N fixation capacity and N cycling processes by reshaping these diazotrophic communities.