AbstractNew, cost-effective source materials are being sought to enable the development of essential oils for potential use in pharmaceutical and commercial applications. The present study was aimed at investigating such features of Cupressus sempervirens, which despite its exotic legendary attributes has been almost ignored up until now. Gas liquid chromatography (GLC) was used to determine the various physico-chemical composition parameters (specific gravity, refractive index, acid and ester values profile) of the selected lignin-containing plant. The essential oil was extracted by a steam distillation technique using needles and twigs of C. sempervirens. The antimicrobial properties of the oil were investigated against a wide spectrum of microorganisms by flask culture and diffusion methods. In the flask culture method, only three strains, viz. A. niger, A. flavous, and A. fumigates, while in diffusion method seven strains, viz. A. niger, A. flavous, A. fumigatous, F. solani, F. oxysporum, Penecillium digitatum, and Candida uterus, and three strains of bacteria, viz. E. coli, M. leutius, B. lacto, exhibited 100% effectiveness in the presence of newly extracted C. sempervirens oil.