Introduction: Herbal products are beneficial compounds with many applications in human life. In this study the chemical composition and cytotoxic activity of the essential oil of the aerial parts of Dorema aucheri were assessed. Methods: The essential oil was extracted by hydrodistillation after drying the aerial parts of D. aucheri, collected from the mountains around Yasuj city in the South-West of Iran. The oil composition was determined by GC/MS. To evaluate in vitro cytotoxic activity, the apoptotic effects of the essential oil were investigated against SW48 and SW1116 colorectal cancer cell lines by (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium) bromide (MTT) assay and flow cytometry. Results: The essential oil yield was obtained 0.02% (W/W). Twenty-five compounds were identified in the oil, and the main constituents were caryophyllene (E) (31.29%), Phytol (14.92%), gurjunene (β-) (9.84%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (8.7%), and n-hexadecanoic acid (8.09%). The MTT assay showed that the IC50 values of the essential oil for SW48 and SW1116 cell lines were 1.4 and 1.2 mg/mL, respectively. The results of flow cytometry showed that the essential oil significantly increased the apoptosis in SW48 cell line compared with the vincristine (P < 0.05). It also increased the apoptosis in SW1116 cells compared with the vincristine, but this difference is not significant. Conclusion: The essential oil of D. aucheri consisted of high amounts of caryophyllene and showed significant cytotoxic effects against SW48 and SW1116 cancerous cell lines.