The development of new high-throughput cultivation methods aims to increase the isolation efficiency as compared to standard techniques that often require enrichment procedures to compensate the low microbial recovery. In the current study, estuarine sulfate-reducing bacteria were isolated using an anaerobic isolation procedure in 384-well microplates. Ninety-nine strains were recovered from initial sediments. Isolates were identified according to their partial 16S rRNA sequences and clustered into 13 phylotypes. Besides, the increase in species richness obtained through enrichments or resampling was investigated. Forty-four enrichment procedures were conducted and shifts in sulfate-reducing bacterial communities were investigated through dsrAB gene fingerprinting. Despite efforts in conducting numerous enrichment conditions only few of them were statistically different from initial sample. The cultural diversity obtained from 3 of the most divergent enrichments, as well as from resampled sediments equally contributed to raise the sulfate-reducing diversity up to 22 phylotypes. Enrichments (selection of metabolism) or resampling (transient populations and micro-heterogeneity) may still be helpful to assess new microbial phylotypes. Nevertheless, all the newly cultivated strains were all representatives of minor Operational Taxonomic Units and could eventually be recovered by maintaining high-throughput isolation effort from the initial sediments.