Background: Currently, many compounds of different medicinal plants are under investigation for curing various types of cancers. In extension with this ongoing research, Arnebia euchroma is taken for the study of cytotoxic compounds present in its roots. The sensitivity of human cervical cancer (SiHa), human epidermoid carcinoma (KB), and human colorectal cancer 116 (HCT116) cell lines toward C1 and C2 isolated from A. euchroma roots was evaluated. Materials and Methods: The root extract of A. euchroma was subjected to extraction, chromatographic separation to afford two naphthazarin esters C1 and C2 as well as two white-colored compounds. The structures of purified compounds were determined by electrospray ionization mass spectrometry and nuclear magnetic resonance analysis. Results: Both C1 and C2 showed potent cytotoxicity against SiHa, KB, HCT116 cells at different concentrations. Conclusion: C1 and C2 exhibited strong cytotoxicity with IC50= 0.02 μM and 0.09 μM against SiHa cells and with IC50= 0.11 μM HCT116 cells than the vinblastine standard, respectively.