Introduction: Zanthoxylum rhetsa is a tree found in northern Thailand. It has been used as a traditional herb with medicinal properties. However, the comparison of composition in volatile oil from fresh and dried fruit is still not fully investigated. In study, we evaluated the constituents in oil from fruits of Z. rhetsa and evaluated the cytotoxicity in non-small lung cancer cells. Methods: The hydro distillation was applied to afford the oil. The obtained oil was analyzed by GC-MS and evaluated the cytotoxicity and apoptosis in non-small lung cells by using MTT method and flow cytometry respectively. Results: Fresh and dried fruits provided a higher yield of volatile oil by approximately 10% and 20% respectively, compared to other studies. Twenty-eight compounds were identified and the major components of fresh and dried fruits were not distinctly different. The major component, α-limonene, was found in dried fruits from Phayao, southern Nan and Chiang Rai province. A high content of β-phellandrene was found in dried fruits from northern Nan province and the (+)-sabinene was found in high content of fresh fruits from southern Nan, Phayao and Chaing Rai province. Furthermore, the cytotoxicity tests displayed that all of oil products were active against lung cancer cells. Among the components investigated, fresh and dried fruits from southern Nan province showed to be the most potent (EC50 = 1.91 ± 0.53, 1.79 ± 0.43 μL/mL, respectively). Conclusion: Volatile oil of Z. rhetsa exhibited a strong cytotoxic properties against cytotoxic may be potentially used as natural anticancer agents.