Origanum onites L. belongs to the Lamiaceae family which is commonly used for symptomatic treatment of the diseasesof the gastrointestinal, endocrine, and respiratory systems in Turkish ethnobotanical studies and public. In this study our aim was toinvestigate the physiological effects of O. onites essential oil (OE) on A549 human lung cancer, MIA PaCa human pancreas cancer andHUVEC human endothelial cell lines. Our findings showed that O. onites OE treatment at all concentrations (4.79–191.6 µg/mL, 24 h)can inhibit cell proliferation and induce apoptosis in A549 and MIA PaCa cell lines (P < 0.0001). Although ≤19.16 µg/mL OE treatmentfor 24 h did not inhibit cell proliferation in the HUVEC cell lines, higher concentrations of OE (≥38.32 µg/mL) significantly inhibitedcell proliferation. Apoptotic cell counts were significantly (P < 0.0001) higher in all cell lines at both incubation times (24 h 56.3 ± 7%,48 h 53 ± 5% for A549, 24 h 44 ± 7%, 48 h 44.7 ± 7% for MIA PaCa, 24 h 48.7 ± 6%, 48 h 49.3 ± 5% for HUVEC). Migration rates weredecreased significantly (P < 0.0001) (24 h 64 ± 6%, 48 h 67.2 ± 4% for A549, 24 h 49 ± 5%, 48 h 69.35 ± 5% for MIA PaCa, 24 h 47 ± 5%,48 h 69.6 ± 2% for HUVEC). Based on these results, OE has been shown to have promising anticancer effects.