The essential oil of fresh aerial parts of Hypericum richeri subsp. grisebachii obtained by hydrodistillation was analyzed by gas chromatography (GC) and gas chromatography/mass spectral GC/MS. One hundred and five constituents identified accounted for 96.1% of the total oil. The major components of the oil were: germacrene D (13.1%), dodecanal (11.9%), beta-caryophyllene (7.4%), beta-pinene (5.8%), caryophyllene oxide (4.2%) and (E)-beta-ocimene (3.3%). The volatile profile of H. richeri subsp. grisebachii was characterized by a large amount of sesquiterpenoids (56.2%), especially by hydrocarbons of this fraction (34.1%). The in vitro antimicrobial activity of essential oil was also examined against a panel of microbial strains by broth microdilution assay and it was found to have moderate effect against all tested microorganisms.