Caves are considered to be an extreme environment for life. However, closer investigation might reveal that life in caves can be unexpectedly various. Calcite moonmilk, a secondary cave deposit was examined for bacterial component in our study. The origin of this material is still discussed although earlier works showed occurence of microorganisms and the possibility of their influence on formation of moonmilk. Active or passive role of microbiota is still unclear. The aim of our work was to characterize isolated bacterial strains by using a polyphasic taxonomic approach and also find an equivalence of bacterial phenotypic characteristics according to a place of sampling. Moonmilk was sampled in caves of The Moravian Karst, Czech Republic and also in caves in Slovakia. 82 bacterial strains were isolated from the moonmilk samples on four different media. The samples were aerobically cultivated at 10 and 15 degrees of Celsia. The isolated psychrotrophic bacterial strains were characterized by biochemical, physiological tests and whole-cell protein analysis by SDS-PAGE. Selected group of strains was also characterized by ribotyping and MALDI-MS TOF. Partial 16S rRNA gene sequencing was performed on 10 gram-negative strains and followed by sequence alignment analysis with the GenBank BLAST program. Only two isolates represented gram-positive rods and the rest of strains were gram-negative non-fermenting rods. The majority of them were classified as fluorescent pseudomonads with some atypical biochemical properties. Phenotypic characteristics of some strains even showed equivalence among distant places of sampling.