Varroa bee hive attack is a serious and common problem in bee keeping. In our work, an ectomicroflora of Varroa destructor mites was characterised as a potential source of bacterial bee diseases. Using a cultivation approach, a variable population of bacteria was isolated from the body surface of Varroa mites with frequency of about 150 cfu per mite individual. Nine studied isolates were classified to four genera and six species by a combination of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-and 16S ribosomal RNA (rRNA)-based methods, suggesting relatively low diversity of Varroa mite-associated ecto-microflora. The Varroa mite-associated bacterial population was found to be dominated by Gram-positive bacteria of Bacillus and Microbacterium genera. Gram-negative bacteria were represented by members of Brevundimonas and Rhizobium genera. Most of the identified species are not known to be associated with Varroa mite, either honey bee or honey up until now and some of them are probably representatives of new bacterial taxa. Varroa destructor / ecto-microflora / heterotrophic bacteria / Microbacterium