Bacteria can adversely affect the quality of drainage released from mine waste by catalyzing the oxidation of sulfide minerals and thereby accelerating the release of acidity and metals. However, the microbiological and geochemical controls on drainage quality from unsaturated and geochemically heterogeneous waste rock remain poorly understood. Here, we identified coexisting neutrophilic and acidophilic bacteria in different types of waste rock, indicating that robust endemic consortia are sustained within pore-scale microenvironments. Subsequently, natural weathering was simulated in laboratory column experiments with waste rock that contained either in-situ microbial consortia or suppressed populations with up to 1000 times smaller abundance and reduced phenotypic diversity after heating and drying. Drainage from waste rock with in-situ populations was up to two pH units lower and contained up to 16 times more sulfate and heavy metals compared to drainage from waste rock bearing treated populations, indicating significantly higher sulfide-oxidation rates. The drainage chemistry was further affected by sorption and formation of secondary-mineral phases (e.g., gypsum and hydroxy-carbonates). This study provides direct evidence for the existence of diverse microbial communities in waste rock and their important catalytic role on weathering rates, and illustrates the mutual controls of microbiology and geochemistry on waste-rock drainage quality. Display omitted •Integrated approach to study microbial and geochemical controls on drainage quality.•Characterization of microbial abundance and community structures in waste rock.•Waste-rock weathering under simulated field conditions using column experiments•Demonstration of microbial catalysis of waste-rock weathering•Discrete secondary minerals and adsorption affect waste-rock drainage quality.