BACKGROUND: A possible reduction in stocks of medicinal plant raw materials of Thymus serpyllum L. and Thymus vulgaris L. leads to the need to expand the raw material base of the official medicinal plants with using of endemic species of the flora of Kazakhstan, in particular, Thymus rasitatus Klokov, and Thymus eremita Klokov. AIM: The aim of the study was to study the possibility of using 70% ethanol extracts of T. rasitatus and T. eremita as antimicrobial agents. MATERIALS AND METHODS: The aerial parts of T. rasitatus and T. eremita were extracted with 70% ethanol using ultrasound assisted extraction. The qualitative and quantitative analyses of the 70% ethanol extracts were determined using the liquid chromatography-detection-ESI-mass spectrometry-(MS)/MS technique. The study of the antimicrobial activity of these extracts was performed for eight strains of Gram-positive bacteria, six strains of Gram-negative bacteria, and four cultures of fungi. RESULTS: Chromatographic analysis of hydroalcoholic extracts of both investigated Thymus species showed very similar phenolic compounds composition. In both cases, the major components are luteolin-7-O-glucoside and rosmarinic acid. About 70% ethanol extracts of T. rasitatus and T. eremita have a broad spectrum of antimicrobial activity, exhibit the bactericidal or bacteriostatic activity against all tested bacteria and fungi at concentration range of 0.0195–20 mg/ml, but differ in their potency against tested strains of microorganisms. CONCLUSION: About 70% ethanol extracts of T. rasitatus and T. eremita, endemic plants in the flora of Kazakhstan, can be considered as potential drugs with a wide spectrum of antimicrobial activity. The results of chromatographic analysis could be used for drug standardization.