Herpes simplex virus Type 1 (HSV-1) causes a wide spectrum of diseases in humans, including skin and mucosal ulcers, encephalitis, and keratitis. Acyclovir is regarded as the gold standard for treating infections with this virus. However, there are certain drawbacks to using this drug, such as its ineffectiveness against treatment-resistant virus strains. Therefore, the development of novel and effective drugs to combat this virus is urgently needed. The present work aims to explore the efficacy of magnesium oxide nanoparticles (MgONPs) against HSV-1 in vitro as a potential novel antiviral agent. MgONPs were characterized by X-ray diffraction, energy-dispersive X-ray spectroscopy, field-emission scanning electron microscope, ultraviolet-visible spectrophotometry, Fourier-transform infrared spectroscopy, dynamic light scattering, and zeta potential. To assess the cytotoxic effects of MgONPs on Vero cells, the neutral red uptake assay was used. The effects of MgONPs at nontoxic concentrations on HSV-1 were then examined using a quantitative real-time PCR assay. No toxic effect was observed in all used concentrations of MgONPs (up to a concentration of 1000 μg/mL). Three-hour incubation of HSV-1 with MgONPs at concentrations of 900 and 1000 μg/mL resulted in a remarkable decrease in viral load with an inhibition rate of 93.6% and 96.8%, respectively. The results from the posttreatment assay also showed that MgONPs at concentrations of 300 and 1000 μg/mL led to a significant decrease in viral load with an inhibition rate of 99.5% and 99.7%, respectively. MgONPs can exert their inhibitory effects on HSV-1 in a dose-dependent manner, both directly and through interfering with the replication cycle of the virus.