Improving the knowledge on sulfate-reducing bacteria (SRB) diversity and ecophysiology will permit a better understanding on their key roles in aquatic ecosystems. Therefore, their diversity was evaluated in estuarine sediments by a polyphasic approach including dsrA gene cloning and sequencing (156 clones) and high-throughput isolations in 384-well microplates (177 strains). Using the related thresholds of 95% (DsrA amino acid sequences) and 97% (16S rRNA gene sequences) for sequence similarity, SRB were grouped into 60 and 22 operational taxonomic units, respectively. Both approaches poorly overlapped and rather complemented each other. The clone library was dominated by sequences related to the Desulfobacteraceae, while only one isolate belonged to this family. Most of the strains were affiliated to the genera Desulfopila and Desulfotalea within the Desulfobulbaceae. Desulfopila-related strains exhibited a high phylogenetic microdiversity and represented numerically significant populations. In contrast, Desulfovibrio isolates were less abundant but displayed a high phylogenetic diversity. Three hundred and eighty-four-well microplate isolations enhanced significantly the number of isolates handled. As a consequence, 15 new taxa sharing less than 98% sequence similarity (16S rRNA gene) with their closest relatives were obtained. This polyphasic approach allowed to obtain a high phylogenetic diversity and thus a better view of sulfate-reducing communities in intertidal sediments. © 2012 Federation of European Microbiological Societies.