The indigenous microbial communities of two extremely acidic, metal-rich stratified pit lakes, located in the Iberian Pyrite Belt (Spain), were identified, and their roles in mediating transformations of carbon, iron, and sulfur were confirmed. A combined cultivation-based and culture-independent approach was used to elucidate microbial communities at different depths and to examine the physiologies of isolates, which included representatives of at least one novel genus and several species of acidophilic Bacteria. Phosphate availability correlated with redox transformations of iron, and this (rather than solar radiation) dictated where primary production was concentrated. Carbon fixed and released as organic compounds by acidophilic phototrophs acted as electron donors for acidophilic heterotrophic prokaryotes, many of which catalyzed the dissimilatory reduction in ferric iron; the ferrous iron generated was re-oxidized by chemolithotrophic acidophiles. Bacteria that catalyze redox transformations of sulfur were also identified, although these Bacteria appeared to be less abundant than the iron oxidizers/reducers. Primary production and microbial numbers were greatest, and biogeochemical transformation of carbon, iron, and sulfur, most intense, within a zone of c. 8–10 m depth, close to the chemocline, in both pit lakes. Archaea detected in sediments included two Thaumarchaeota clones, indicating that members of this recently described phylum can inhabit extremely acidic environments. Keywords