Introduction Osteosarcoma (OS) is a primary bone sarcoma with a high recurrence rate and poorer prognosis. The application of natural agents in combinational therapies can increase the efficacy of treatment and decrease the side effects. Herein, we aimed to evaluate the effects of Thymoquinone (TQ) combined with Cisplatin on apoptosis and its underlying mechanisms in the Saos-2 cells. Methods The effects of TQ and Cisplatin on Saos-2 cell viability were measured using an MTT assay. Western blotting was applied for the measurement of γH2AX protein expression. The expression levels of 8-Hydroxy-2'-deoxyguanosine (8-oxo-dG) were evaluated by enzyme-linked immunosorbent assay (ELISA). DCFH-DA fluorescence dye was used to detect reactive oxygen species (ROS) formation. For evaluation of apoptosis, flow cytometry was employed. Results TQ dramatically promotes the cytotoxic effects of Cisplatin. TQ considerably enhanced the expression levels of 8-oxo-dG and γ-H2AX in Saos-2 cells. After TQ treatment, ROS levels were increased; furthermore, TQ treatment resulted in the potentiation of Cisplatin-induced apoptosis in Saos-2 cells compared to either TQ or Cisplatin treated cells. Conclusion In general, TQ plus Cisplatin resulted in potentiated cellular cytotoxicity by increasing ROS level and inducing oxidative DNA damage, leading to the potent induction of apoptosis in tumor cells.