Background: Depth of invasion (DOI) significantly influences prognosis and treatment strategies in oral squamous cell carcinoma (OSCC). Accurate preoperative imaging, such as contrast-enhanced computed tomography (CECT), alongside postoperative histopathological evaluations, aids in determining DOI. This study evaluates the correlation between radiological DOI (rDOI), macroscopic DOI (PDOI), and microscopic DOI (pDOI) in OSCC. Methods: This study included 54 OSCC patients from April 2022 to November 2023. rDOI was assessed using preoperative CECT, while PDOI and pDOI were measured through histopathological examination of resected specimens. Spearman correlation analysis and Bland-Altman plots assessed agreement between DOI measurements, with statistical significance set at p < 0.05. Results: Strong correlations were found between rDOI and PDOI (r = 0.713), rDOI and pDOI (r = 0.688), and PDOI and pDOI (r = 0.897, p < 0.001 for all). CECT overestimated DOI in T1 and T2 lesions, particularly in ulcerative tumors. Bland-Altman analysis showed mean differences of 1.86 mm (rDOI-PDOI) and 3.3 mm (rDOIpDOI). Higher correlations were observed in the presence of perineural invasion (PNI), lymphovascular invasion (LVI), and worst pattern of invasion 5 (WPOI 5), with r values up to 0.948 (rDOI-PDOI) and 0.980 (PDOI-pDOI). Conclusion: While rDOI correlates strongly with pathological DOI, overestimations in smaller and ulcerative lesions necessitate cautious interpretation. Pathological risk factors, including PNI, LVI, and WPOI 5, were associated with greater DOI and enhanced agreement between radiological and pathological assessments. Overall, CECT is a reliable tool for preoperative evaluation of DOI.