Little is known about the long-term impacts of metal contamination on the microbiota of anoxic lake sediments. In this study, we examined microbial biomass and metals (arsenic, cadmium, chromium, copper, iron, lead, manganese, and zinc) in the sediments of Lake DePue, a backwater lake located near a former zinc smelter. Sediment core samples were examined using two independent measures for microbial biomass (total microscopic counts and total phospholipid phosphate concentrations) and for various fractions of each metal (pore water extracts, sequential extractions, and total extracts of all studied metals and zinc speciation by X-ray absorption fine structure). Zinc concentrations were up to 1000 times higher than reported for sediments in the adjacent Illinois River and ranged from 21,400 mg/kg near the source to 1,680 mg/kg near the river. However, solid metal fractions were not well correlated with pore water concentrations and were not good predictors of biomass concentrations. Instead, biomass, which varied among sites by as much as 2 times, was inversely correlated with concentrations of pore water zinc and arsenic as established by multiple linear regression. Monitoring of other parameters known to naturally influence biomass in sediments (e.g., organic carbon concentrations, nitrogen concentrations, pH, sediment texture, and macrophytes) revealed no differences that could explain observed biomass trends. This study provides strong support for control of microbial abundance by pore water metal concentrations in contaminated freshwater sediments.