Three acidophilic bacteria were isolated from water and sediment samples collected at a coal mining-impacted river, Rio Sangão, located in the city of Criciúma, Santa Catarina state, in south Brazil. These microorganisms were isolated in acid media and were phylogenetic related to the species Acidiphilium cryptum by its 16s rRNA gene sequences, although they differ from this species in the assimilation of some carbon sources. The optimum and the maximum pH for growth of all strains were nearly 3.0 and 5.0-6.0, respectively. Two of the strains were slightly more acidophilic, with the minimum pH for growth of 2.0. All strains also tolerate the four tested metals (Ni, Zn, Cu and Se) at variable concentrations, with LAMA 1486 being the most metal-resistant strain. These bacteria may belong to different ecotypes of A. cryptum, or even represent new species in the genus. Besides they bear characteristics that make them useful in the development of bioremediation process, for the treatment of sites contaminated with multiple toxic metals, including coal mining drainage