Background: Nasopharyngeal carcinoma (NPC) is a head and neck cancer more frequent among East Asian populations compared with Western populations. While much is known about human papillomavirus’s (HPV’s) role in oropharyngeal cancer (OPC), little is known about its prevalence and prognostic value in NPC. The aim of this study is to investigate the role of HPV in NPC treated with definitive radiotherapy at a single institution. Methods: A retrospective cohort analysis of patient’s medical records and HPV status treated for NPC in Rambam Health Care Campus (Rambam HCC). Immunohistochemical staining for p16 was used as a surrogate marker of HPV infection in the tumor cells. All specimens were stained and evaluated by pathologists at the referring center independently. Results: In total, 87 patients diagnosed with NPC were treated at Rambam HCC between 2005 and 2018. Seventy-four patients had accessible data on the disease’s clinical parameters and p16 status. In total, 10/74 (13.5%) had p16-positive staining in tumor cells; 75% were men and over 50% were smokers. The average age of diagnosis for the whole cohort was 48 years, being lower for p16-positive patients compared with p16-negative patients at 43 and 49 years old, respectively. A total of 84% of the patients had advanced disease of stage III and IV at presentation. Only 16% were diagnosed with stage I and II. Unlike the p16-negative group, the p16-positive group did not include any stage I or II disease. In univariate and multivariate analysis of overall survival rates, the age at diagnosis and the nodal spread status were the only statistically significant measures. P16 status was not found to be associated with survival. Conclusions: The HPV prevalence in NPC is nontrivial. p16-positive patients had significantly less nodal spread and tended to be younger. Both age and nodal status were significantly correlated with the survival, but P16 status was not prognostic. Further large-scale trials are needed to elucidate the role of HPV in NPC.