There are several sources of mine drainage left over from past mining sites in the Central Slovakian neovolcanites. The neutral pH and high concentrations of sulphates and multiple potentially hazardous elements, such as zinc or manganese, are typical in this region. However, this environment could be home to specific microbiota. The aim of the study was to characterize bacterial populations in mine drainage in the Central Slovakian neovolcanites. Direct microscopic observations, cultivation methods, MALDI TOF mass spectrometry, and 16S rRNA gene sequencing of isolates were used for identification. Gram-positive and Gram-negative bacteria were almost equally represented in the mine water samples. The most abundant bacterium was the genus Bacillus spp. (43.48%). Another large group of bacteria consisted of Proteobacteria (34.78%), represented by Pseudomonas spp. (17.39%), Serratia spp. (13.04%), and Providencia spp. (4.35%). Our data confirm the presence of Bacillus spp. and Pseudomonas spp. as bacterial species occurring in an environment polluted by potentially hazardous elements, which may indicate their bioremediation potential.