In the present study, essential oil extracted by hydrodistillation from Gladiolus atroviolaceus Boiss. (Iridaceae) was analyzed by gas chromatography (GC) and GC-mass spectrometry (MS). A total of 52 compounds were identified, constituting over 93.8 % of the oil composition of G. atroviolaceus. Hexadecanoic acid (44.4 %) and heneicosane (6.6 %) were the major compounds in the volatile of G. atroviolaceus. Sesquiterpene hydrocarbons were shown to be the main groups of terpenoids in the oils (2.6 %). The major terpene constituents of the essential oil of G. atroviolaceus were limonene, gamma-muurolene and linalool. The antimicrobial activity of the isolated essential oil of the plant was also investigated and it showed moderate antimicrobial and antifungal activities against Staphylococcus aureus, Bacillus cereus, Mycobacterium smegmatis and Candida albicans.