In situ characterization and geochemical modelling of acid generation in a mine tailings lake (Moose Lake, ON, Canada) over a 2‐year period (2001–2002; surficial lake pyrrhotite slurry disposal initiated in 2002) show that bacteria significantly impact acidity behaviour through particle‐associated S oxidation and that they do so under conditions that differ from those controlling abiotic pathways. Seasonal epilimnetic pH decreases occurred in both years, decreasing from approximately 3.5 in May to 2.8 by September (2001) or July (2002). Epilimnetic acid generation rates were depth‐dependent, with maximal rates observed not at the surface of lake where O2 concentrations were highest, but rather within a geochemically reactive zone (approximately 1 m thick) of steep, decreasing O2 gradients and dynamic Fe and S geochemistry in the lower epilimnetic region of the lake. Acid generation occurred dominantly through particle rather than aqueous pathways, but model predictions of acid generation via abiotic pyrrhotite oxidation involving either O2 or ferric iron (Fe3+) predicted neither the observed rates nor the depths at which maximal rates occurred. In contrast, model predictions based on microbial pathways involving both O2 and ferric iron (Fe3+) agreed extremely well with both the observed depth profile of H+ generation and the observed rates at any given depth. Imaging showed extensive microbial colonization of epilimnetic‐associated pyrrhotite particles commonly with significant biofilm formation. FISH (fluorescence in situ hybridization) probing of the community in both pelagic and particle compartments indicated mixed communities occurred in both, and that Acidithiobacillus spp. accounted for 2–46% of the total community in either compartment. Initiation of pyrrhotite slurry discharge at the lake surface in 2002 was accompanied by a relative increase in the number of particle‐associated microbes, as well as a relative proportional decrease of Acidithiobacillus spp. in the total microbial community. Given the widespread occurrence of bacteria across mining environments, the implications of our results extend beyond this specific site and provide new insight into bacterially driven processes contributing to bulk system characteristics which are not currently well constrained.