The essential oil was obtained by hydrodistillation from the aerial parts of Origanum rotundifolium Boiss. Its chemical content and composition were analyzed by using a gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Total phenolic content of the essential oil was determined as 132.39 mu g gallic acid equivalent by Folin-Ciocalteu's method and the major component was identified as carvacrol (56.8 %) along with p-cymene (13.1 %), (Z)-beta-ocimene (5.4 %), beta-caryophyllene (3.9 %), borneol (3.4 %) and thymol (3.2 %). After chemical characterization, the essential oil was evaluated for its antioxidant activity by DPPH free radical, superoxide anion radical and hydrogen peroxide scavenging activities as well as ferrous ion-chelating power test, ABTS radical cation decolorization assay and ferric thiocyanate methods. Besides antioxidant activity, acetylcholinesterase and butyrylcholinesterase inhibitory activities of the essential oil were also evaluated by Ellman's method. It demonstrated inhibitory activities on AChE and BuChE, key enzymes in the pathogenesis of Alzheimer's disease (AD), in addition to significant antioxidant activity.