High resolution analysis of collagen bundles could provide information on tumor onset and evolution. This study was focused on the microarchitecture and biomolecular organization of collagen bundles in oral tongue squamous cell carcinoma (OTSCC). Thirty-five OTSCC biopsy samples were analyzed by synchrotron-based phase-contrast microcomputed tomography and Fourier transform infrared imaging (FTIRI) spectroscopy. PhC-microCT evidenced the presence of reduced and disorganized collagen in the tumor area compared to the extratumoral (ExtraT) one. FTIRI also revealed a reduction of folded secondary structures in the tumor area, and highlighted differences in the peritumoral (PeriT) areas in relation with the OTSCC stage, whereby a significantly lower amount of collagen with less organized fibers was found in the PeriT stroma of advanced-OTSCC stages. Interestingly, no significant morphometrical mismatches were detected in the same region by PhC-microCT analysis. These results suggest that biomolecular alterations in the OTSCC stroma temporally anticipate structural modifications of collagen bundle microarchitecture.