Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor with a poor patient prognosis. Standard of care treatment includes maximal surgical resection, followed by radiotherapy and temozolomide administration. Despite this multimodal strategy, patients fail to achieve an effective response due to resistance to radio-treatment, allowing the tumor to recur. Therefore, there is a compelling need to discover new radiosensitizer approaches to overcome radioresistance and improve the response and survival of patients affected by glioblastoma. A novel approach is represented by nanoradiosensitizers, such as gold nanoparticles (AuNPs), able to enhance the effectiveness of radiotherapy. In this study, we assessed the biological interaction of AuNPs with ionizing radiation (IR) in both immortalized and primary patient-derived glioblastoma cell lines. we observed that the combinatory effect of AuNPs with IR decreased cell viability and increased necrosis and apoptosis compared to cells treated only with IR or untreated cells. Additionally, our results showed an increase in the sensitization enhancement ratio (SER) of cells treated with AuNPs and IR. Furthermore, AuNPs showed a tumor-specific effect, since it did not seem to support the effects of radiation on the normal human astrocyte (NHA) cell line. Our results suggest that the use of AuNPrs can improve radiotherapy efficacy by increasing the radiosensitivity of the targeted cells.