The mass-independent minor oxygen isotope compositions (Δ′17O) of atmospheric O₂ and CO₂ are primarily regulated by their relative partial pressures, pO₂/pCO₂. Pyrite oxidation during chemical weathering on land consumes O₂ and generates sulfate that is carried to the ocean by rivers. The Δ′17O values of marine sulfate deposits have thus been proposed to quantitatively track ancient atmospheric conditions. This proxy assumes direct O₂ incorporation into terrestrial pyrite oxidation-derived sulfate, but a mechanistic understanding of pyrite oxidation—including oxygen sources—in weathering environments remains elusive. To address this issue, we present sulfate source estimates and Δ′17O measurements from modern rivers transecting the Annapurna Himalaya, Nepal. Sulfate in high-elevation headwaters is quantitatively sourced by pyrite oxidation, but resulting Δ′17O values imply no direct tropospheric O₂ incorporation. Rather, our results necessitate incorporation of oxygen atoms from alternative, 17O-enriched sources such as reactive oxygen species. Sulfate Δ′17O decreases significantly when moving into warm, low-elevation tributaries draining the same bedrock lithology. We interpret this to reflect overprinting of the pyrite oxidation-derived Δ′17O anomaly by microbial sulfate reduction and reoxidation, consistent with previously described major sulfur and oxygen isotope relationships. The geologic application of sulfate 017O as a proxy for past pO₂/pCO₂ should consider both 1) alternative oxygen sources during pyrite oxidation and 2) secondary overprinting by microbial recycling.