Objectives: The aim of this study was to investigate the cytotoxic and radical scavenging effects of the essential oil from Origanum acutidens (Hand-Mazz.) Ietswaart and to determine its chemical composition. Materials and Methods: The essential oil was obtained by hydrodistillation and analyzed by gas chromatography/mass spectrometry (GC-MS). The cytotoxic effect of the essential oil on the growth of human colorectal adenocarcinoma (HT-29) and human cervical adenocarcinoma (HeLa) cell lines was investigated by xCELLigence Real Time Cell Analyzer instrument. Moreover, radical scavenging effect of the oil was determined by using DPPH method. Results: The main constituents of the oil were found to be carvacrol (61.69%), p-cymene (17.32%), γ-terpinene (4.05%), and borneol (3.96%). The essential oil of O. acutidens exhibited significant cytotoxic effect against HeLa and HT-29 cell lines at the tested concentrations. The essential oil had moderate DPPH radical scavenging activity compared to butylated hydroxytoluene (BHT). Conclusion: The data in the present study clearly demonstrated inhibitory effect of the oil on two human cancer cell lines. According to these results and other reported studies, this observed high effect may be attributed to the presence of the carvacrol component in the oil.