We have explored the sulfur metabolism and accompanying fractionation of sulfur isotopes during the disproportionation of elemental sulfur by seven different enrichments and three pure bacterial cultures. Cultures were obtained from both marine and freshwater environments. In all cases appreciable fractionation accompanied elemental sulfur disproportionation, with two ranges of fractionation observed. All cultures except Desulfobulbus propionicus produced sulfide depleted in 34S by between 5.5 and 6.9 per mil (avg of 6.3 per ml) and sulfate enriched in 34S by between 17.1 and 20.2 per mil (avg of 18.8 per ml). The narrow range of fractionations suggests a conserved biochemistry for the disproportionation of elemental sulfur by many different marine and freshwater bacteria. Fractionations accompanying elemental sulfur disproportionation by Db. propionicus were nearly twice as great as the others, suggesting a different cellular level pathway of sulfur processing by this organism. In nearly every case pyrite formation accompanied the disproportionation of elemental sulfur. By using sulfur isotopes as a tracer of sulfur source, we could identify that pyrite formed both by the addition of elemental sulfur to FeS and from reaction between FeS and H.S. Both processes were equally fast and up to 104−105 times faster than expected from the reported kinetics of inorganic pyrite‐formation reactions. We speculate that bacteria may have enhanced rates of pyrite formation in our experimental systems. The organisms explored here have different strategies for growth and survival, and they may be active in environments ranging from dissolved sulfide‐poor suboxic sediments to interfaces supporting steep opposing gradients of oxygen and sulfide. A large environmental range, combined with high bacterial numbers, significant isotope fractionations, and a possible role in pyrite formation, make elemental sulfur‐disproportionating bacteria potentially significant actors in the sedimentary cycling of sulfur compounds.