We studied the response of the sulfate-reducing prokaryote (SRP) communities to the experimental variation of salinity and tide in an outdoor mesocosm setup. Intact soil monoliths were collected at two areas of the Haringvliet lagoon (The Netherlands): one sampling location consisted of agricultural grassland, drained and fertilized for at least the last century; the other of a freshwater marshland with more recent sea influence. Two factors, i.e., “salinity” (freshwater/oligohaline) and “tide” (nontidal/tidal), were tested in a full-factorial design. Soil samples were collected after 5 months (June–October). Dissimilatory (bi)sulfite reductase β subunit-based denaturing gradient gel electrophoresis (dsrB-DGGE) analysis revealed that the SRP community composition in the agricultural grassland and in the freshwater marshland was represented mainly by microorganisms related to the Desulfobulbaceae and the Desulfobacteraceae, respectively. Desulfovibrio-related dsrB were detected only in the tidal treatments; Desulfomonile-related dsrB occurrence was related to the presence of oligohaline conditions. Treatments did have an effect on the overall SRP community composition of both soils, but not on the sulfate depletion rates in sulfate-amended anoxic slurry incubations. However, initiation of sulfate reduction upon sulfate addition was clearly different between the two soils.