Chinese medicine is renowned worldwide. Researchers have devoted their efforts to isolating the functional ingredients of Traditional Chinese Medicine preparations to develop new medications for tumor treatment. Traditional cinobufotalin (CBF) preparations are effective herbal ingredients isolated from toad venom and are widely used for the treatment of inflammation, to induce anesthesia, and treat cardiac arrhythmias. Natural bufaledienolides, such as cinobufagin (CB) and CBF, are cardiotonic steroids with a chemical structure and physiological activity analogous to those of digoxin. Previous studies have mainly focused on their cardiac properties; however, in the last decade, the broad-spectrum antitumor activity of these natural bufaledienolides has received increasing attention. In particular, they have been shown to exert significant antitumor effects by inhibiting tumor cell proliferation, metastasis, tumor stemness, lipogenesis, and by promoting apoptosis. Moreover, chemically synthesized CB and CBF have shown promising anticancer activity and application prospects. In this review, we summarized the involvement of CB and CBF in clinical applications, their associated adverse effects, and their regulatory activity on signaling networks in tumors. In addition, we also summarized the feasibility of a nanoparticle-mediated drug delivery system for improving the effectiveness of CB and CBF as antitumor therapy.