Background: Oncolytic viruses (OVs) have been utilized since 1990s for targeted cancer treatment. Our study examined the Measles–Mumps–Rubella (MMR) vaccine’s cancer-killing potency against Glioblastoma (GBM), a therapy-resistant, aggressive cancer type. Methodology: We used GBM cell lines, primary GBM cells, and normal mice microglial cells, to assess the MMR vaccine’s efficacy through cell viability, cell cycle analysis, intracellular viral load via RT-PCR, and Transmission Electron Microscopy (TEM). Results: After 72 h of MMR treatment, GBM cell lines and primary GBM cells exhibited significant viability reduction compared to untreated cells. Conversely, normal microglial cells showed only minor changes in viability and morphology. Intracellular viral load tests indicated GBM cells’ increased sensitivity to MMR viruses compared to normal cells. The cell cycle study also revealed measles and mumps viruses’ crucial role in cytopathic effects, with the rubella virus causing cell cycle arrest. Conclusion: Herein the reported results demonstrate the anti-cancer activity of the MMR vaccine against GBM cells. Accordingly, the MMR vaccine warrants further study as a potential new tool for GBM therapy and relapse prevention. Therapeutic potential of the MMR vaccine has been found to be promising in earlier studies as well.