Talimogene laherparepvec (T-VEC), an oncolytic herpes simplex virus, is approved for intralesional injection of unresectable stage IIIB/IVM1a melanoma. However, it is still unclear which parameter(s) predict treatment response or failure. Our study aimed at characterizing surface receptors Nectin-1 and the herpes virus entry mediator (HVEM) in addition to intracellular molecules cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) as potential bio-markers for oncolytic virus treatment. In 20 melanoma cell lines, oncolytic activity of T-VEC was correlated with the expression of Nectin-1 but not HVEM, as evaluated via flow cytometry and immunohistochemistry. Knockout using CRISPR/Cas9 technology confirmed the superior role of Nectin-1 over HVEM for entry and oncolytic activity of T-VEC. Neither cGAS nor STING as evaluated by Western Blot and immunohistochemistry correlated with T-VEC induced oncolysis. The role of these biomarkers was retrospectively analyzed for the response of 35 cutaneous melanoma metastases of 21 patients to intralesional T-VEC injection, with 21 (60.0%) of these lesions responding with complete (n = 16) or partial regression (n = 5). Nectin-1 expression in pretreatment biopsies significantly predicted treatment outcome, while the expression of HVEM, cGAS, and STING was not prognostic. Altogether, Nectin-1 served as biomarker for T-VEC-induced melanoma regression in vitro and in vivo.