© 2016 Elsevier B.V.. The aim of this study was to investigate volatile composition of the essential oil (EO) of Nepeta sintenisii Bornm. (N. sintenisiig), and its antioxidant, antimicrobial and cytotoxic activities. The EO was obtained by means of hydrodistillation and its components were analyzed using gas chromatography and mass spectrometry (GC/MS). The main constituents of the EO were found to be 4aα,7α,7aβ-Nepetalactone (51.74%), β-Farnesene (12.26%), 4aα,7α,7aα-Nepetalactone (8.01%), Germacrene-D (5.01%), and 4aα,7β,7aα-Nepetalactone (3.71%); these together constituted 95.81% of the extracted oil. The antimicrobial activity of the EO against eleven bacterial strains and one fungus was studied. The EO showed potent and broad-spectrum antibacterial effects against both Gram-positive and Gram-negative bacteria, and also possessed antifungal activity against Candida albicans. In vitro cytotoxicity evaluation against four cell lines of human ovarian carcinoma (A2780), cervical cancer (Hela), human colon adenocarcinoma (LS180), and human breast adenocarcinoma (MCF-7), and Human Umbilical Vein Endothelial Cells (HUVEC (as a normal cell line), showed a potent anti-proliferative activity of the oil with the lowest IC50 value (20.37 μg/mL) belonging to the Hela cells. The IC50 value for normal human cell line (HUVEC) was 219 μg/mL. Also, antioxidant activity of the EO was evaluated using 1,1-diphenyl-2-picrilhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods. The oil exhibited weak antioxidant activity in DPPH test (IC50: 7.16 mg/mL) and FRAP (IC50: 0.82 mM Fe2+/mg EO) assay. The present results suggested promising cytotoxic, antibacterial, and antifungal properties for the N. sintenisii EO.