Sulfide petrography plus whole rock contents and isotope ratios of sulfur were measured in a 1.5 km section of oceanic gabbros in order to understand the geochemistry of sulfur cycling during low-temperature seawater alteration of the lower oceanic crust, and to test whether microbial effects may be present. Most samples have low SO4/ΣS values (≤ 0.15), have retained igneous globules of pyrrhotite ± chalcopyrite ± pentlandite, and host secondary aggregates of pyrrhotite and pyrite laths in smectite ± iron-oxyhydroxide ± magnetite ± calcite pseudomorphs of olivine and clinopyroxene. Compared to fresh gabbro containing 100–1800 ppm sulfur our data indicate an overall addition of sulfide to the lower crust. Selection of samples altered only at temperatures ≤ 110 °C constrains microbial sulfate reduction as the only viable mechanism for the observed sulfide addition, which may have been enabled by the production of H2 from oxidation of associated olivine and pyroxene. The wide range in δ34Ssulfide values (− 1.5 to + 16.3‰) and variable additions of sulfide are explained by variable εsulfate-sulfide under open system pathways, with a possible progression into closed system pathways. Some samples underwent oxidation related to seawater penetration along permeable fault horizons and have lost sulfur, have high SO4/ΣS (≥ 0.46) and variable δ34Ssulfide (0.7 to 16.9‰). Negative δ34Ssulfate–δ34Ssulfide values for the majority of samples indicate kinetic isotope fractionation during oxidation of sulfide minerals. Depth trends in sulfide–sulfur contents and sulfide mineral assemblages indicate a late-stage downward penetration of seawater into the lower 1 km of Hole 735B. Our results show that under appropriate temperature conditions, a subsurface biosphere can persist in the lower oceanic crust and alter its geochemistry.