Rock, air and service water samples were collected for microbial analyses from 3.2 kilometres depth in a working Au mine in the Witwatersrand basin, South Africa. The ∼ metre-wide mined zone was comprised of a carbonaceous, quartz, sulphide, uraninite and Au bearing layer, called the Carbon Leader, sandwiched by quartzite and conglomerate.The microbial community in the service water was dominated by mesophilic aerobic and anaerobic, α-, β- and γ-Proteobacteria with a total biomass concentration ∼104 cells ml-1, whereas, that of the mine air was dominated by members of the Chlorobi and Bacteroidetes groups and a fungal component.The microorganisms in the Carbon Leader were predominantly mesophilic, aerobic heterotrophic, nitrate reducing and methylotrophic, β-and γ-Proteobacteria that were more closely related to service water microorganisms than to air microbes. Rhodamine WT dye and fluorescent microspheres employed as contaminant tracers, however, indicated that service water contamination of most of the rock samples was <0.01% during acquisition. The microbial contaminants most likely originated from the service water, infiltrated the low permeability rock through and accumulated within mining-induced fractures where they survived for several days before being mined. Combined PLFA and terminal restriction fragment length profile (T-RFLP) analyses suggest that the maximum concentration of indigenous microorganisms in the Carbon Leader was <102 cells g-1. PLFA, 35S autoradiography and enrichments suggest that the adjacent quartzite was less contaminated and contained ∼103 cells gram-1 of thermophilic, sulphate reducing bacteria, SRB, some of which are δ-Proteobacteria. Pore water and rock geochemical analyses suggest that these SRB s may have been sustained by sulphate diffusing from the adjacent U-rich, Carbon Leader where it was formed by radiolysis of sulphide.