Knowledge on the molecular and clinical characteristics of head and neck squamous cell carcinoma (HNSCC) is vast. However, an effective therapy that increases the life expectancy of these patients, with a 5-year overall survival of 50%, is still unknown. Here we evaluated the combined effect of the pro-apoptotic protein rhTRAIL with the replication-competent wild-type HSV-1 virus in head and neck cancer cell lines. We observed a difference in the modulation profile of proteins related to apoptotic pathways in the studied cell lines. The HCB289 exhibited caspase-9 activation in the presence of the HSV-1 virus, while the UD-SCC-2 exhibited caspase-8 activation in the presence of rhTRAIL. Both cell lines exhibited PARP activation by combining rhTRAIL and HSV-1 virus treatment. Flow cytometry analysis exhibited greater induction of late apoptosis for the HCB289 and UD-SCC-2 after the combination treatment of the HSV-1 and rhTRAIL. However, the UD-SCC-2 also presented induction of late apoptosis by the presence of rhTRAIL in monotherapy. These data suggest an enhancement of the effect of the combination treatment of the rhTRAIL and the HSV-1 on reducing viability and induction of cell death.