The O, S, and Sr isotope compositions were determined for 17 samples of gypsum that replaced anhydrite in the sheeted-dike complex of the Macquarie Island ophiolite. Elevated δ34S (26.2‰–29.0‰) and δ18O values (12.5‰–14.4‰) of gypsum compared to those of seawater sulfate are the result of microbial sulfate reduction. Low organic carbon contents and little sulfate reduction in sediments, plus a large basaltic Sr component in the gypsum (87Sr/86Sr = 0.70446–0.70524), indicate that the sulfate source was not pore waters in the overlying sediment. Low δ34S values of sulfide in basalt lavas are consistent with microbial reduction of seawater sulfate within the volcanic rocks. Tectonic activity at the slow-spreading ridge allowed evolved formation waters to enter hot sheeted-dike complex basement, resulting in heating and precipitation of anhydrite. Results show that microbes can leave geochemical tracers of their activity in oceanic basement and that anhydrite can be preserved in oceanic crust and may be of significance for the oceanic sulfur budget.