The aim of this study was to investigate the diffusion dynamics of 25% hydrogen peroxide (H₂O₂) through enamel-dentin layers and to correlate it with dentin’s structural alterations. Micro-Raman Spectroscopy (MRS) and Fourier Transform Infrared Photoacoustic Spectroscopy (FTIR-PAS) were used to measure the spectra of specimens before and during the bleaching procedure. H₂O₂ was applied to the outer surface of human enamel specimens for 60 minutes. MRS measurements were performed on the inner surface of enamel or on the subsurface dentin. In addition, H₂O₂ diffusion dynamics from outer enamel to dentin, passing through the dentin-enamel junction (DEJ) was obtained with Raman transverse scans. FTIR-PAS spectra were collected on the outer dentin. MRS findings revealed that H₂O₂ (O-O stretching µ-Raman band) crossed enamel, had a more marked concentration at DEJ, and accumulated in dentin. FTIR-PAS analysis showed that H₂O₂ modified dentin’s organic compounds, observed by the decrease in amides I, II, and III absorption band intensities. In conclusion, H₂O₂ penetration was demonstrated to be not merely a physical passage through enamel interprismatic spaces into the dentinal tubules. H₂O₂ diffusion dynamics presented a concentration gradient determined by the chemical affinity of the H₂O₂ with each specific dental tissue.