Аннотация:In the present study, the gas-chromatography—mass-spectrometry methods and classic bacteriology were used for investigation of microbiological diversity of underground waters in the Vorotilov deep well (East European platform, at 1500, 1900, 3200, 3800, and 4500 m). Mineralization of underground waters was high, 160–190 g/l. The data on microbial diversity (21–27 species of bacteria depending on fluid permeability) and total number of microorganisms (1–1000 g/ml) were obtained by gas-chromatography-mass-spectrometry analysis of specific biomarkers of microbial cells and by classical bacteriological cultivation on selectve media. Dominant groups among native microorganisms were the genera Clostridium, Corynebacterium, and Rhodococcus. Prevalence of heterotriophic processes was noted. The number of chemolithoautotrophic microorganisms was insignificant, but they increase when there is lower pollution of water. The number of chemolithoautotrophs was low. However, they increased when water pollution was lower (which was shown by classic bacteriology methods). The “detritus” type of the investigated subsurface ecosystem might result from contamination of underground water by a drilling solution. Biomarker monitoring along the column of a deep well reflected the hydrodynamics determined both by geological conditions and exploitation of the well.