Abstract Leakage of crude oil and gas through fault conduits intersecting the seafloor gives rise to scores of point-source anoxic enclaves on the oxic northern Gulf of Mexico slope. A study of 13 short cores recovered with a manned submersible from these seepage-affected sediments reveals that microbial processes fueled by hydrocarbons cause extensive sulfur diagenesis. Sulfate reduction and sulfide release occurring in the pore fluids reach completion 10–25 cm below the sediment–water interface. Bacterial sulfate reduction (BSR) rates are highly variable between sites but maximum values (97 and 917 μmol SO 4 cm −3 year −1) in a bacterial mat and mussel bed, respectively, are unusually high for cold, deepwater habitats. δ 34S and δ 18O values of the residual sulfate range from 20.7‰ to 70.8‰ (CDT) ( n=45) and from 11.1‰ to 23.6‰ (SMOW) ( n=33) compared to the overlying Gulf of Mexico bottom water values of 20.3‰ and 9.7‰, respectively. δ 34S values of H 2S yield a mean of 12.4±5.4‰ (CDT) ( n=15). δ 34S (SO 4) data yield an integrated fractionation factor of α S=1.015 under a closed system assumption but a substantial higher fractionation of α S=1.023 under an open system assumption. Paired SO 4–H 2S inventory indicates that up to 28% of sulfide is removed from the system and supports the contention that seep sediments constitute an open system. The sulfur isotope fractionations reported here compare well with experimental data for cold-adapted sulfate-reducing bacteria but are substantially smaller than the “geological” fractionation of α S=1.055 derived from coeval sulfate-sulfide in Phanerozoic sediments. Isotope enrichments in the SO 4 are 2.4 times greater in δ 34S than in δ 18O and the relations documented with f(SO 4) are indicative of mixing between two end-member sulfate sources; seawater sulfate cycled through microbial dissimilatory sulfate reduction at depth and secondary sulfate produced by oxidation of H 2S at near-surface through bacterial disproportionation (BDS) processes. The evidence for superimposed metabolic reactions in the reducing and oxidative parts of the anaerobic sulfur cycle in seep sediments has important implications regarding the proposed use of pore-water sulfate profiles as proxies of upward methane fluxes resulting from dissociation of marine-based gas hydrates.