Origanum vulgare L., an aromatic and medicinal plant has been used extensively for the food and pharmaceutical industry. In this work, the essential oil was generated by steam distillation and the compounds were identified by GC-MS analysis. Carvacrol was found as a major product. O. vulgare was extracted with water (60 °C) to yield the water extract as well. The antiproliferative activity and cytotoxic effect of essential oil and extract were investigated using MTT [3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyl tetrazolium bromide] and LDH (Lactate dehydrogenase) techniques, respectively. A549 (human lung carcinoma), Hep3B (hepatocellular carcinoma), HT29 (human colon carcinoma), MCF7 (human breast adenocarcinoma) cancerous cell lines, FL (human amnion cells) normal cell lines were used for essential oil and extract, while cisplatin and 5-FU were exploited as standards. The essential oil revealed the significant activity against A549 (IC50, 27.2 µg/mL), Hep3B (IC50, 7.4 µg/mL), and MCF-7 (IC50, 7.1 µg/mL) cancerous cell lines as compared to standards. Moreover, extract displayed outstanding activity against Hep3B (IC50, 27.2 µg/mL) and MCF-7 (IC50, 10.8 µg/mL) cell lines. The activity of essential oil may be due to carvacrol since carvacrol is the main constituent of essential oil with a high percentage (90.4%) or due to the synergistic effect of the compounds in the essential oil.