In this study, volatile compounds (VCs) in the essential oil (EO), SPME and SPME of n-hexane extract of Vinca major subsp. hirsuta (Boiss.) Stearn were analyzed by GC-FID/MS instrument. A total of 32, 38, and 26 compounds with in the 98.5%, 98.6%, and 98.6% were identified, respectively. As a result of VCs study, (Z)-3-hexenol (36.8%) in the EO, 1,3,5-trimethylbenzene (31,5%) in the SPME, and phenylethyl alcohol (32.8 %) in the SPME of n-hexane extract of V. major subsp. hirsuta were found to be major compounds. Sesquiterpenes (20.6%, and 11.5%) for the HD and SPME, monoterpenes (18.6%) for the SPME of n-hexane extract of V. major subsp. hirsuta were found as the main groups among the terpenic compounds, respectively. Then, the antimicrobial activity of the EO and the solvent extracts (n-hexane, acetonitrile, methanol, and water) of V. major subs. hirsuta against 3 gram negative, 3 gram positive, 1 tuberculosis and 2 fungus were screened. The EO showed the only activity against the Mycobacterium smegmatis ATCC607 (MIC, 152 µg/mL) and Candida albicans ATCC60193 (MIC, 38 µg/mL). The n-hexane extract did not show any activity against all tested microorganisms. The best antimicrobial activity for the acetonitrile, methanol, and water extracts were observed against M. smegmatis with 69 µg/mL, 609 µg/mL, and 437 µg/mL MIC values, respectively. None of the extracts were found to be active to Enterococcus faecalis ATCC29212, Staphylococcus aureus ATCC25923, and Saccharomyces cerevisiae RSKK 251.