Background: Depth of invasion (DOI) is a key determinant of staging and management in oral cavity squamous cell carcinoma (OSCC), particularly in guiding decisions regarding elective neck dissection. DOI is frequently reported on incisional biopsy specimens pre-operatively. This study aimed to assess the reliability of biopsy-derived DOI in predicting final pathological DOI and its association with nodal disease. Methods: A retrospective cohort study was performed of 118 patients with clinically staged T1–T2 OSCC treated surgically at a tertiary centre between 2012 and 2024. DOI from initial biopsy was compared with DOI from the final surgical specimen. Statistical analysis included paired t-tests and chi-square analysis, with significance set at p < 0.05. Associations with nodal disease were also evaluated. Results: DOI was reported in 73/118 biopsy specimens (62%). The mean DOI on biopsy was 2.6 mm compared to 5.48 mm on final pathology, representing a significant underestimation (p < 0.001). Biopsy DOI resulted in understaging in 17% of cases, with 17% of tumours upstaged to pathological T3/T4 disease. When analysed categorically, 60.3% of tumours were upgraded to a higher DOI group on final pathology, while only 39.7% remained unchanged. Overall, 45% of tumours were upstaged according to AJCC staging criteria. There was poor concordance between biopsy and final DOI groupings (χ² p = 0.003). Notably, even among tumours with a biopsy DOI <2 mm, 20% demonstrated nodal metastasis. Overall nodal disease was present in 19.5% of patients. Conclusion: Biopsy-derived DOI significantly underestimates true tumour invasion and demonstrates poor agreement with final pathology. Given the high rates of upstaging and the presence of nodal disease even in low DOI groups, biopsy DOI should not be used in isolation for clinical decision-making, particularly in determining the need for elective neck dissection