Tomm34, as a member of the outer mitochondrial membrane proteins, is evenly distributed between the cytoplasm and the outer mitochondrial membrane. It is up-regulated in a variety of tumors and correlates with poor prognosis. This study aimed to investigate expression of Tomm34 and its correlations with clinicopathology in oral squamous cell carcinoma (OSCC). Oncomine database and UALCAN database were utilized to predict the expression and prognosis values of Tomm34 in head and neck squamous cell carcinoma (HNSCC). By immunohistochemistry, a retrospective study was performed to verify the bioinformatics results to evaluate the Tomm34 expression and clinicopathological variables in both HPV-positive OSCC and HPV-negative OSCC. Immunohistochemistry of our cohort revealed that 48 cases fulfilled the Tomm34 high expression judgment criteria, and the overall positive rate was 60% (48/80), and 27 cases fulfilled the p16 expression judgment criteria (33.75%, 27/80). The high expression of Tomm34 was closely related with the TNM classification of OSCC (p < 0.01) and tumor size (p < 0.01) both in HPV-negative OSCC and HPV-positive OSCC, while related with lymph node metastasis (p = 0.001) in HPV-negative OSCC and drinking history (p = 0.044) in HPV-positive OSCC. In addition, the Kaplan-Meier curves indicated that higher level of Tomm34 was correlated with poorer overall survival (OS) and disease-free survival (DFS) in HPV-negative OSCC (OS, p = 0.046; DFS, p = 0.020) but not in HPV-positive OSCC (OS, p = 0.824; DFS, p = 0.782). In conclusion, Tomm34 is highly expressed in OSCC and may be a useful factor to provide prognostic information, especially in HPV-negative OSCC group.