Cervical cancer is the fourth most common gynecological malignancy affecting the health of women worldwide and the second most common cause of cancer‑related mortality among women in developing regions. Thus, the development of effective chemotherapeutic drugs for the treatment of cervical cancer has become an important issue in the medical field. The application of natural products for the prevention and treatment of various diseases, particularly cancer, has always attracted widespread attention. In the present study, a library of natural products composed of 78 single compounds was screened and it was found that digitoxin exhibited the highest cytotoxicity against HeLa cervical cancer cells with an IC50 value of 28 nM at 48 h. Furthermore, digitoxin exhibited extensive antitumor activities in a variety of malignant cell lines, including the lung cancer cell line, A549, the hepatoma cell line, MHCC97H, and the colon cancer cell line, HCT116. Mechanistically, digitoxin caused DNA double‑stranded breaks (DSBs), inhibited the cell cycle at the G2/M phase via the ataxia telangiectasia mutated serine/threonine kinase (ATM)/ATM and Rad3‑related serine/threonine kinase (ATR)‑checkpoint kinase (CHK1)/checkpoint kinase 2 (CHK2)‑Cdc25C pathway and ultimately triggered mitochondrial apoptosis, which was characterized by the disruption of Bax/Bcl‑2, the release of cytochrome c and the sequential activation of caspases and poly(ADP‑ribose) polymerase (PARP). In addition, the in vivo anticancer effect of digitoxin was confirmed in HeLa cell xenotransplantation models. On the whole, the findings of the present study demonstrate the efficacy of digitoxin against cervical cancer in vivo and elucidate its molecular mechanisms, including DSBs, cell cycle arrest and mitochondrial apoptosis. These results will contribute to the development of digitoxin as a chemotherapeutic agent in the treatment of cervical cancer.