The aim of this work was to determine the microbial diversity of the acid mine drainage (AMD) material collected at an abandoned pyrite mine in Ouro Preto, Brazil. AMD samples were compared to a nickel sulfide column bioleaching pregnant solution which was used as reference. Fluorescent in situ hybridization analyses (FISH) and Denaturing Gradient Gel Electrophoresis (DGGE) were used. FISH analysis was carried out using specific 16S rRNA probes. The extracted DNA was amplified using universal primers for bacterial 16S rRNA genes and analyzed by DGGE. Acidithiobacillus. ferrooxidans was not detected in AMD samples. However, the presence of Acidithiobacillus thiooxidans was confirmed. In other hand, in the bioleaching tanks samples studied, both bacteria species were detected. The non-identified DNA bands were cloned and sequenced for complete characterization.