Background Fusobacterium nucleatum (F. nucleatum) has been increasingly linked to oral squamous cell carcinoma (OSCC), prompting this study to explore its presence using polymerase chain reaction (PCR) and evaluate its clinical significance. Methods In this pilot case-control study, 12 OSCC tissue samples and 12 non-cancerous oral mucosal tissue samples were analyzed. Total RNA extraction and complementary DNA (cDNA) synthesis were performed using Trizol-based methods, followed by PCR amplification and gel electrophoresis. The clinical characteristics of participants and PCR results were recorded. Results Among the OSCC tissue samples, three out of 12 tested positive for F. nucleatum, while none of the control samples showed its presence. The detection rate of F. nucleatum in OSCC was 25%. Gel analysis confirmed specific amplicon amplification, and ImageJ software enabled copy number quantification. Discussion Our findings support previous research indicating a potential association between F. nucleatum and OSCC. Understanding the etiological significance of F. nucleatum in OSCC has clinical implications, including early detection, risk stratification, and prognostication. However, the limited sample size and the need for further research to elucidate underlying mechanisms are acknowledged. Conclusion This pilot study provides initial evidence of F. nucleatum's presence in a subset of OSCC samples, supporting its potential association with oral cancer. Detecting F. nucleatum in OSCC tissues holds promise for future research and clinical applications as a diagnostic and prognostic biomarker. Understanding its role in oral carcinogenesis will facilitate the development of targeted therapeutic strategies. Larger studies are warranted to validate these findings and investigate the precise mechanisms involved.