Secondary metabolites present in lichens, which comprise aliphatic, cycloaliphatic, aromatic and terpenic compounds, are unique with respect to those of higher plants and show interesting biological and pharmacological activities. However, only a few of these compounds, have been assessed for their effectiveness against various in vitro cancer models. In the present study, we investigated the cytotoxicity of three lichen secondary metabolites (atranorin, gyrophoric acid and physodic acid) on A375 melanoma cancer cell line. The tested compounds arise from different lichen species collected in different areas of Continental and Antarctic Chile. The obtained results confirm the major efficiency of depsidones. In fact, depsides atranorin and gyrophoric acid, showed a lower activity inhibiting the melanoma cancer cells only at more high concentrations. Whereas the depsidone physodic acid, showed a dose-response relationship in the range of 6.25–50 μM concentrations in A375 cells, activating an apoptotic process, that probably involves the reduction of Hsp70 expression. Although the molecular mechanism, by which apoptosis is induced by physodic acid remains unclear, and of course further studies are needed, the results here reported confirm the promising biological properties of depsidone compounds, and may offer a further impulse to the development of analogues with more powerful efficiency against melanoma cells.