An investigation of microbial communities able to form biofilms and inhabiting an extreme acid mine drainage (AMD) polymetallic mine with pH ranging from 1.0 to 1.5 was carried out. Presented results concern an abandoned polymetallic mine that has not been studied so far. Geochemical analyses of the sampled area reveals a high concentration of heavy metals – especially arsenic and iron derived from the decomposition of arsenopyrite. Cryo-SEM analyses of hydrated biofilm reveals its structure and composition, showing intact extracellular polysaccharides (EPS) with minerals submerged in an EPS matrix. Thus a direct connection between bacteria and biotransformation of surrounding minerals can be observed. Microbial community analyses were carried out by using the non-cultivated method based on DNA extraction, cloning, sequencing, and molecular phylogenetics. Bioinformatics analyses reveals the presence of bacteria belonging to three phylogenic groups: Proteobacteria, Acidobacteria, and Actinobacteria. The majority of them were characterized as iron-oxidizing bacteria. The information presented in this work is critical to understand which microorganisms are important to AMD production in the studied area and involved in iron and sulfur cycles.