Drug resistance mechanisms still characterize metastatic melanoma, despite new treatments have been recently developed. Targeting of the cGMP/protein kinase G (PKG) pathway is emerging as a therapeutic approach in cancer research. In this study, we evaluated the anticancer effects of two polymeric linked dimeric (PDL) cGMP analogues able to bind and activate PKG, called PKG-activator (PA) 4 and 5. PA5 was identified as the most effective compound on melanoma cell lines as well as on patient-derived metastatic melanoma cells cultured as three-dimensional spheroids and in a zebrafish melanoma model. PA5 was able to significantly reduce cell viability, size, and invasion of melanoma spheroids. Importantly, PA5 showed a tumor specific outcome because no toxic effect was observed in healthy melanocytes exposed to the cGMP analogue. We defined that, by triggering PKG, PA5 interfered with the EGF pathway as shown by lower EGFR phosphorylation and reduction of activated, phosphorylated forms of AKT and ERK1/2 in melanoma cells. Finally, PA5 significantly reduced the metastatic process in zebrafish. These studies open future perspectives for the cGMP analogue PA5 as a potential therapeutic strategy for melanoma.