Although neutral mine drainage is the less frequent subject of the interest than acid mine drainage, it can have adverse environmental effects caused mainly by precipitation of dissolved Fe. The aim of the study was to characterize the composition of bacterial population in environment with high concentration of iron and sulfur compounds represented by neutral mine drainage water of Elizabeth's shaft, Slovinky (Slovakia). Direct microscopic observations, cultivation methods, and 454 pyrosequencing of the 16S rRNA gene amplicons were used to examine the bacterial population. Microscopic observations identified iron-oxidizing Proteobacteria of the genera Gallionella and Leptothrix which occurrence was not changed during the years 2008-2014. Using 454 pyrosequencing, there were identified members of 204 bacterial genera that belonged to 25 phyla. Proteobacteria (69.55%), followed by Chloroflexi (10.31%) and Actinobacteria (4.24%) dominated the bacterial community. Genera Azotobacter (24.52%) and Pseudomonas (14.15%), followed by iron-oxidizing Proteobacteria Dechloromonas (11%) and Methyloversatilis (8.53%) were most abundant within bacterial community. Typical sulfur bacteria were detected with lower frequency, e.g., Desulfobacteraceae (0.25%), Desulfovibrionaceae (0.16%), or Desulfobulbaceae (0.11%). Our data indicate that the composition of bacterial community of the Elizabeth's shaft drainage water reflects observed neutral pH, high level of iron and sulfur ions in this aquatic habitat.