Introduction Alternative medicine is important in cancer treatment. The apoptotic effect of Thymol and extracted Thymol from Thymbra spicata on non-small-cells lung cancer was studied. Material and methods Thymol was evaluated in Thymbra spicata extract by HPCL. Cell viability was assessed by MTT method. DCF and flu3-AM probe was used for ROS and cai2+ analysis, respectively. Western blotting was performed to measure NOX2 and Bax/Bcl-2 ratio. Results Obtained data showed that Thymol was 1.51 mg/g in Thymbra spicata extract. Treatment with Thymol and extracted Thymol from Thymbra spicata resulted in cell death at high concentrations [LC50= 111±4.5 and 119±5.2 μM, respectively]. Subsequently, Thymbra spicata extract and its bioactive component increased ROS and Cai2+ production, NOX2, and Bax/Bcl-2 ratio. Conclusions This study revealed the anticancer effects of Thymol and Thymbra spicata extract on non-small-cells lung cancer and at least part of that effect was related to the increase in the NOX2 and Bax/Bcl-2 ratio. Our results demonstrated that TSE and Thymol at high concentrations (180, 120, and 80 μM) decreased the growth of A549 cells. It appeared that cytotoxic activity was exerted through activation of NOX2, ROS generation, increase in Cai2+, and Bax/Bcl-2 ratio. Present results demonstrated that TSE and thymol may be potential therapeutic agents for human lung cancer.