BACKGROUND Human papillomavirus (HPV) and Epstein-Barr virus (EBV) are associated with head and neck cancer, including tonsil cancer (TC) in the oropharyngeal area. Increasing incidence of HPV and EBV infection in different cancer tissues of oropharynx in both epithelial and lymphoid tissues, have been reported. However, little is known about association of these tumor viruses with TC in the Thai population. Here, we investigated the prevalence of HPV and EBV infection in different histology of TC and their association with TC from Thai patients. METHODS Eighty-three exfoliated tonsil cells from non-cancer controls (NCC) and 65 formalin-fixed paraffin-embedded TC tissues (TC) that were histologically classified as tonsillar squamous-cell carcinoma (TSCC) or diffuse large B-cell lymphoma (DLBCL) were studied. Prevalence of HPV and EBV infection was determined by real-time PCR. HPV genotyping was performed by reverse line blot hybridization and HPV genome status was investigated by multiplex qPCR. Localization of EBV infection was determined by EBER in situ hybridization. RESULTS Infection of HPV and EBV in TC cases was 16.9% and 30.8%, whereas in exfoliated tonsil cells was 1.2% and 66.3% respectively. HPV infection was significantly higher in TSCC (30.6%) than DLBCL samples (13.8%). HPV58 was commonly detected and presented as an integrated form in TSCC, whereas only episomal form was found in DLBCL. EBV infection was significantly higher in DLBCL (44.8%) than TSCC samples (19.4%), and detected in both lower than among exfoliated tonsil cell samples (66.3%). By EBER in situ hybridization in TSCC, EBV infection localized both in epithelial cells and infiltrating lymphocytes. The co-occurrence of HPV and EBV infection was 11.11% and 13.79% of TSCC and DLBCL, respectively, was associated with well-differentiated TSCC. CONCLUSIONS HPV and EBV infection was significantly involved in a specific TC tissue, and associated with a good clinical outcome in TSCC.