The inclusion of nanoparticles (NP) in radiotherapy has been shown to increase the damaging effect on tumor cells. However, the mechanisms of action of NP combined with radiotherapy, and the influence of NP parameters and cell type on their radiosensitization capability at molecular and cellular levels still remain unclear. Gold NP (AuNP) have become particularly popular due to their multiple advantages. Within this context, our research work aimed to study the biochemical radiosensitization capacity of F98 and U87-MG glioma cell lines to 1.9 nm AuNP combined with X-ray irradiation. For this purpose, synchrotron-based infrared microspectroscopy (SR-FTIRM) was used as a powerful tool for biochemical composition and treatment response assessment of cells at a single-cell level. SR-FTIRM data, supported by multivariate analysis, revealed clear AuNP-induced changes in the DNA, protein and lipid spectral regions. The AuNP-related biochemical alterations appear prior to the irradiation, which gave us a first indication on the AuNP radiosensitization action. Biochemical modifications induced by the AuNP in the presence of radiotherapy irradiations include enhanced conformational changes in the protein secondary structures, variations in the intensity and position in the phosphodiester bands, and changes in the CH2 and CH3 stretching modes. These changes are better manifested at 24 hours post-irradiation time. SR-FTIRM results showed a clear heterogeneity in the biochemical cell response, probably due to the distinct cell-NP interactions and thus, to different DNA damage and cell death processes.