Macrophomina phaseolina, a necrotrophic fungus with global distribution, has emerged as a source of bioactive natural products. This study evaluated the antiviral potential of metabolites from M. phaseolina against herpes simplex virus 1 (HSV-1). A crude extract of M. phaseolina was obtained from the culture medium, and the secreted metabolites were purified using chromatographic techniques. Major metabolites were identified using NMR and HRMS techniques. The antiviral activity of these metabolites was assessed using Vero cells and virus quantitation through qPCR. In addition, a viral gene expression and intracellular genome quantification was performed using qPCR. Six metabolites were isolated: (−)-botryodiplodin, mellein, hydroxymellein, acetylphomalactone, asperlin, and isoasperlin. Their structures were elucidated through NMR and HRMS. Most metabolites showed limited antiviral efficacy; however, (−)-botryodiplodin demonstrated potent activity, with an EC₅₀ of 2.44 µg/mL and a selectivity index of 4.79. In addition, acyclovir was included as a control, demonstrating >97% antiviral inhibition. Mechanistic studies showed that (−)-botryodiplodin inhibited HSV-1 production, reduced viral genome levels, and suppressed immediate–early, early, and late viral gene expressions. These findings demonstrate that (−)-botryodiplodin acts as an early-stage inhibitor of HSV-1 prior to viral transcription, establishing this natural metabolite as a promising chemical scaffold for the rational design of novel antiviral agents.