Bone samples extracted from embalmed cadavers are commonly used as controls in the study of bone. The effects of embalmment on the molecular composition of bone are unknown. The objective of this study was to determine the effect of embalmment on the molecular composition and structure of bone, as evaluated by Raman spectroscopy. Bone samples of femoral heads from five embalmed donors and five fresh-frozen donors were compared using Raman microspectroscopy with DuoScan technology. Physicochemical parameters simultaneously describing the organic and mineral phases of bone were compared using the Mann–Whitney U test. Partial least squares discriminant analysis (PLS-DA) was used to determine specific Raman spectral features of each group. Study of the mineral phase showed a 15% reduction of the mineral-to-matrix ratio (p < 0.001), an 8% decrease of type B carbonate substitution (p < 0.001), and a 2% increase in crystallinity (p < 0.001) in the embalmed donors group compared to those of the fresh donors group. Regarding the organic phase of bone, the hydroxyproline-to-proline ratio was increased by 18% in the embalmed group (p < 0.001), with no variation in both the relative proteoglycan content (GAG/CH3) (p = 0.08) and collagen maturity (p = 0.57). PLS-DA showed that the embalmed group was characterized mainly by peaks assigned to hydroxyproline, lipids, and collagen. Embalmment induces significant modifications of the molecular composition of bone. Bone samples from embalmed subjects should be avoided as controls for Raman spectroscopy studies. Preservation procedures performed prior to bone sampling should be reported in studies using human cadaver samples.