Centaurea species are used in eastern Mediterranean ethnopharmacology due to variety of bioactive compounds they comprise. Aim of this work was to characterize the Centaurea rupestris L. hydrodistilled essential oil chemical composition and test its biological activity: antimicrobial effect, antioxidant potential and inhibition of cholinesterases. Plant material authentication was by chromosome number counting and genome size assessment with the flow cytometry. Hydrodistilled essential oils were characterized using gas chromatography/mass spectrometry technique GC-MS and GC-FID. The antimicrobial effect was tested using disk diffusion and microdilution methods, antioxidant potential was tested with DPPH and FRAP methods and cholinesterases inhibition was tested with Ellman method. Genome size for C. rupestris species: sample A presented 2C=3.60 (0.10) pg and sample B 2C=3.62 (0.08) pg. The chromosome number was 2n=20 for both samples. The main essential oil constituents in isolated sample A oil, detected with GC-MS and GC-FID were: germacrene D (24.3 %), heptacosane (14.4 %), phytol (6.7 %), β-caryophyllene (5.0 %) and pentacosane (4.5 %). Sample B essential oil had the main constituents: hexadecanoic acid (18.7 %), heptacosane (13.8 %), α-linolenic acid (11.8 %), nonacosane (7.8 %) and germacrene D (5.4 %). Both samples of oil showed broad spectrum antimicrobial effect with good activity against emerging Gram-positive and Gram-negative opportunistic pathogens and pathogenic fungi which indicates the pharmaceutical potential of the C. rupestris essential oil.