Simple Summary Talimogene laherparepvec (T-VEC), a first-in-class oncolytic herpes simplex virus, improves the outcome of patients suffering from unresectable melanoma, in particular in combination with checkpoint inhibitors. However, a certain percentage of patients does not profit from this treatment, which raises the question of potential biomarkers to predict success or failure of oncolytic herpes viruses. For these purposes, we studied the oncolytic activity of T-VEC in a panel of 20 melanoma cell lines and evaluated the clinical response of 35 melanoma metastases to intralesional T-VEC application. Through these studies, we characterized Nectin-1 as a suitable biomarker predicting 86% and 78% of melanoma regression in vitro and in vivo, respectively. In contrast, other molecules involved in the entry (HVEM) and signal transduction (cGAS, STING) of herpes simplex viruses were not predictive. Altogether, our data support the role of Nectin-1 in pretreatment biopsies to guide clinical decision-making in malignant melanoma and supposedly other tumor entities. 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.