Background: An adequate OSCC macroscopic resection margin (MRM) is essential for effective treatment. This study analyzed the effects of formalin fixation (FF) on the MRM. Material and Methods: A total of 42 patients were enrolled in this study. Tumors from the floor of the mouth (FOM; n = 23), the tongue (TC; n = 10), and the maxilla (MT; n = 9) were studied. A 3D scanner was used to create virtual models, and further analysis was conducted according to the established protocol. Results: The most significant shrinkage was observed in the TC (MRM n = 121; Med. = 1.5 mm; p val. = 7.05 × 10−18), with a maximum shrinkage of 28%. For the FOM (n = 262; Med. = 0.8 mm; p val. = 6.76 × 10−18), the greatest MRM shrinkage was 26%. In the MT group (n = 91; Med. = 0.9 mm; p val. = 2.69 × 10−9), the shrinkage was 18.7%. Among MRMs >8 mm (n = 159), FF led to 58.5% of them shrinking to ≤8 mm, resulting in a false decrease in the safe MRM (p val. = 1.11 × 10−27). Overall, the average shrinkage for all specimens was μ= 2.57 mm (p val. = 8.89 × 10−10) alongside and μ= 2.35 mm (p val. = 4.09 × 10−6) across. The tumors themselves showed minimal changes: μ= 0.69 mm (p val. = 9.73 × 10−3) alongside and μ= 0.8 mm (p val. = 2.52 × 10−7) across. Conclusion: Formalin fixation (FF) caused the shrinkage of the OSCC MRM, particularly in tongue cancers. Even after proper surgical excision in the postoperative results, the number of normal MRMs was underestimated. This should be considered when interpreting the results of surgical treatment. However, FF had a minimal impact on the overall shrinkage of the tumors themselves.