This study was aimed at evaluating the potential application of benzophenanthridine alkaloids, sanguinarine and cheleritrine, in the therapy of melanoma cancer. In vitro antiproliferative activity of sanguinarine was higher than that of cheleritrine against the B16 melanoma 4A5 cells. Both agents were able to produce DNA breaks, and the DNA unwinding assay showed that they act as DNA intercalating agents. Sanguinarine was selected for determination of its in vivo preclinical efficacy. Oral treatment with sanguinarine reduced the tumor burden in a transplantable murine tumor grown in a syngeneic host (B16 melanoma 4A5 in C57BL/6 mice), and in a human tumor xenograft grown in immunodeficient mice (A375 human melanoma in athymic nude mice). In A375 tumors a significant decrease in the proliferation marker Ki67, and a reduction in the activated mitogen-activated protein kinases (p-p44/42 MAPK), and in protein kinase B (pAKT) were also observed. Three out of eleven A375-bearing treated mice were tumor-free at the end of treatment, and did not develop any tumor after a further, treatment-free, observation period of 60 days. Sanguinarine also showed a striking antiangiogenic activity in mice. Data from the present study support the concept that sanguinarine can be effective in melanoma skin cancer.