The radical scavenging, antimicrobial, and antispasmodic activities and the composition of Origanum acutidens (Hand.-Mazz.) Ietswaart (Order Labiatte) essential oil were evaluated in vitro. Gas chromatography/mass spectrometry analysis of the oil resulted in the identification of 20 compounds, representing 93.61% of the oil; carvacrol (65%) was the main component. The sample was also subjected to a screening for antioxidant activity by using the 1,1-diphenyl-2-picrylhydrazyl and beta-carotene/linoleic acid assays. The essential oil exhibited strong antioxidant activity. Using the agar disk diffusion method, antimicrobial activities of the essential oils were determined for nine microorganisms; antimicrobial activity was shown against eight of them. O. acutidens oil exhibited spontaneous contractions in rat ileum at the 100% level at 0.1 mg/mL level.