Background: Esophageal cancer is the eighth most common cancer and the sixth most frequent cancer-related death. Despite improvements in treatment approaches, it has remained one of the most challenging cancers for treatment. Thus, it is necessary to introduce novel and efficient methods for treatment. In this regard, the anti-cancer and antioxidant properties of pomegranate have been more concerned. Objectives: The present study aimed to assess the potential cytotoxicity effects of pomegranate on the esophageal cancer cell line. Methods: We cultured esophageal cancer cell line (KYSE-30) and fibroblast cell line (HF2FF) as normal cell line in RPMI-1640/Ham's F12 and RPMI-1640 medium, respectively, with different concentrations of pomegranate seed oil (2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/mL). Then, we evaluated the cytotoxic effects of pomegranate seed oil via morphologic observation and MTT assay after 24, 48, and 72 hours. The wound-healing assay was used for the evaluation of the mobility and migration potential of the treated cells. Results: The results of the MTT assay showed the cytotoxicity and growth inhibitory effects of pomegranate seed oil on esophageal cancer cells. The viability of tumor cells was significantly reduced compared with the untreated control cells and normal cells (P < 0.05), which were dose and time-dependent. The results of the wound-healing assay indicated that pomegranate seed oil could reduce the migration ability of KYSE-30 cells. Conclusions: According to the results, pomegranate seed oil seems to have cytotoxicity effects on esophageal cancer cells. Thus, it might be considered a new, cheap, and safe treatment option for esophageal cancer.