The light stable isotope chemistry of barite and acanthite in the Pierina Au-Ag deposit, Peru, provides strong support for microbial involvement at the economically critical supergene oxidation stage. Early hypogene barite yields δ34S values of 23.6‰ to 28.5‰ and δ18O values of 5.8‰ to 10.9‰, and was precipitated with the original precious metal-enriched sulfide assemblage. In contrast, late barite, deposited during formation of supergene goethite and hematite, the main ore-hosts, yields δ34S and δ18O values of 1.4‰ to 14.2‰ and −2.8‰ to 4.7‰, correlated mutually and with the δ34S values of coprecipitated acanthite (δ34S = 0.4‰ to 3.9‰). Low δ34S values of barite and acanthite record sulfide mineral oxidation by meteoric waters. Increasing δ34S and δ18O values of barite and δ34S values of acanthite indicate enrichment of the sulfate reservoir in 34S and 18O, reflecting the preferential utilization of light isotopes by microbes during aqueous sulfate reduction. Continued sulfate reduction locally reduced supergene fluids, remobilizing iron and locally destroying the goethite-hematite assemblage. Precious metals were released but reacted with sulfide in the reduced waters to form Au-rich acanthite.