The antioxidant and antimicrobial activity of Costus woodsonii was previously investigated in our lab. The present study investigated the cytotoxic activity of different plant parts and to identify the bioactive compound(s). MTT assay showed that among different plant parts, rhizome extract exhibited the strongest activity (IC50 of 48 hr treatment: <75.8 µg/ml) against all cell lines tested. Using C18 column chromatography, the isolated compound from rhizome, which demonstrated potent cytotoxic activity (IC50 ≤ 2.4 ± 0.2 µg/ml) against oral squamous cell carcinoma was shown to be dioscin by HPLC, LCMS-MS, and NMR analyses. Our findings also showed that dioscin could be a potential treatment for cisplatin-resistant OSCC and C. woodsonii could serve as a potential commercial source for dioscin. Practical applications: Different plant parts of Costus woodsonii have long been used traditionally for medicinal application in water decoction. Nonetheless, the bioactivity study on this plant is scarce. In order to exploit different plant parts of C. woodsonii more scientifically and efficiently for commercial purpose, the present study serves as an important work in understanding the cytotoxic activity of each different parts. This study also provides insight on the effect of extraction solvent on the cytotoxic potential of the selected plant parts. Sequential solvent extraction of rhizome extract followed by fractionation revealed the presence of dioscin, an industrially important steroidal saponin. Overall, this study demonstrated that the rhizome of C. woodsonii may act as a potential source of dioscin.