The present study highlights the biological effects of chromomycin A2 toward metastatic melanoma cells in culture. Besides chromomycin A2, chromomycin A3 and demethylchromomycin A2 were also identified from the extract derived from Streptomyces sp., recovered from Paracuru Beach, located in the northeast region of Brazil. The cytotoxic activity of chromomycin A2 was evaluated across a panel of human tumor cell lines, which found IC50 values in the nM-range for exposures of 48 and 72 h. MALME-3M, a metastatic melanoma cell line, showed the highest sensitivity to chromomycin A2 after 48h incubation, and was chosen as a model to investigate this potent cytotoxic effect. Treatment with chromomycin A2 at 30 nM reduced cell proliferation, but had no significant effect upon cell viability. Additionally, chromomycin A2 induced accumulation of cells in G0/G1 phase of the cell cycle, with consequent reduction of S and G2/M and unbalanced expression of cyclins. Chromomycin A2 treated cells depicted several cellular fragments resembling autophagosomes and increased expression of proteins LC3-A and LC3-B. Moreover, exposure to chromomycin A2 also induced the appearance of acidic vacuolar organelles in treated cells. These features combined are suggestive of the induction of autophagy promoted by chromomycin A2, a feature not previously described for chromomycins.