In recent decades, viral outbreaks have significantly threatened global health, with herpes simplex virus type 2 (HSV-2) being one of the most prevalent infections. This study evaluated novel spiropyrimidine derivatives as potential antiviral agents against HSV-2, building on previous research that examined spirocyclic thiopyrimidinone derivatives against human coronavirus 229E (hCoV-229E). Among the eleven synthesized compounds, spiropyrimidinone derivative 3 demonstrated promising antiviral activity, with a selectivity index of 11.2. The drug mechanism of infection studies indicated that compound 3 primarily inhibits HSV-2 at the viral adsorption stage, achieving approximately 83% inhibition and reducing viral multiplication by 34%. Its efficacy is linked to its diketone moiety, which is known for its ability to enhance antiviral effects. Furthermore, the effect of compound 3 on viral inhibition is reflected in the level of caspase-3 protein expression, revealing that the apoptotic pathway is modulated. Docking studies revealed multiple interactions with herpes virus entry mediator (HVEM), indicating its potential as an entry inhibitor. These findings confirm that compound 3 could be a potential candidate for further development in HSV-2 antiviral therapy.