Río Tinto (Huelva, Spain) is an extreme environment whose origin is a natural underground bioreactor in which the high concentration of metallic sulfides of the Iberian Pyrite Belt are dissolved by microbial activity. As part of a drilling project conducted in the source area of the river, several microorganisms were isolated under strict anaerobic conditions from the deep subsurface of this ecosystem. Here, we report the genomic analysis of these isolates to assess their role in the bioreactor.