Coexisting dissolved sulfide and sulfate from hypersulfidic interstitial waters of a 380-m-long sediment core show a large isotopic difference of up to 72 per mil caused by in situ microbial sulfate reduction. This is considerably larger than the assumed biological maximum of 46 per mil derived from laboratory studies with pure cultures of sulfate-reducing bacteria, so far. Similar high fractionations inferred from sedimentary metal sulfides have been previously explained by a multistage process, involving sulfide reoxidation and disproportionation of sulfur intermediates. Our data suggests that extreme isotopic differences between sulfate and the reduced sulfur species can also be generated during microbial single-step fractionation. This result indicates that the sulfate-reducing communities and/or their cellular metabolic activities in the deep biosphere may differ from those observed in near-surface sediments or the water column.