Pigmented biofilm material from the Homestake gold mine in Lead, South Dakota, USA was examined for chemoheterotrophic culturable microbial isolates. Ten isolates were obtained. The two fastest growing isolates were a Pseudonocardia spp., and a Bacillus thuringiensis isolate. Of the culturable isolates, the Pseudonocardia isolate was best able to grow in iron-limited conditions, suggesting it produced a siderophore. It also had the greatest carboxymethylcellulase activity of any isolate, and appeared to fix dinitrogen. It produced a reddish brown pigment that was the color of the biofilm it was obtained from. The fast-growing B. thuringiensis isolate reduced anthraquinone-2,6- disulfonic acid disodium salt (AQDS) noticeably more than the other isolates. Combined with this information, microscopic evidence suggested that the Pseudonocardia may be a major component of the biofilm, helping provide a physical framework for the other strains to grow in. The B. thuringiensis strain may be important in reducing iron in the iron-rich environment of the biofilm.