To investigate the enzyme-inhibitory efficacy and the cytotoxicity of reticulol produced from a strain of Streptoverticillium, we conducted a DNA topoisomerase (Topo) cleavage assay and an in vivo assay using B16F10 melanoma. From the inhibition assay of reticulol for Topo I, which is involved in melanoma metastasis, it was seen that Topo I treated with 45 μM reticulol did not replicate or transcribe DNA by forming supercoiled DNA. In the annexin V/propidium iodide staining assay to investigate the death pattern of B16F10 cells treated with 200 μM reticulol, proliferation of B16F10 cells was inhibited due to necrosis. Furthermore, from the in vivo assay, reticulol combined with Adriamycin (a mixture with retinolol 5 mg/kg and Adriamycin 1 mg/kg) further retarded the tumor growth compared to that in mice treated with Adriamycin alone (1 mg/kg). The survival rate of tumor-bearing mice treated with the mixture was closely associated with its cytotoxicity. Taken together, these results suggested that reticulol inactivates Topo I, which is involved in tumor metastasis, and exhibits excellent cytotoxic efficacy against B16F10 melanoma, when combined with Adriamycin, in a mouse model.