Human Papillomavirus (HPV) has emerged as a significant etiological factor in various epithelial malignancies, including oral squamous cell carcinoma (OSCC). This case-control study aimed to explore the association of HPV with OSCC and to analyze the histopathological features correlating with its presence. 120 individuals, comprising 60 OSCC cases and 60 matched controls, were enrolled. HPV DNA was detected using polymerase chain reaction (PCR), and histopathological evaluation was performed to assess cellular atypia, keratinization patterns, and lymphovascular invasion. The results revealed a statistically significant higher prevalence of HPV in OSCC cases compared to controls (p < 0.001), with HPV16 being the predominant genotype. Histopathological analysis demonstrated pronounced cellular atypia and increased mitotic figures in HPV-positive OSCC cases (mean atypia index: 3.6 ± 1.1) compared to HPV-negative cases (mean atypia index: 2.2 ± 0.9, p = 0.004). The findings suggest a potential oncogenic role of HPV in OSCC pathogenesis, emphasizing its prognostic significance This study bridges the gap in understanding the HPV-associated histopathological alterations in OSCC, providing valuable insights into its pathobiology. The results underscore the need for routine HPV screening in OSCC patients and advocate for targeted therapeutic strategies.