Herpes simplex virus type 1 (HSV-1) is a persistent human pathogen, and the emergence of strains resistant to Acyclovir (ACV, reference drug) shows the urgency to develop new treatments. We report the antiherpetic mechanism of the action of lasiodiplodan (LAS-N, (1 → 6)-β-d-glucan) and its sulfonated derivative (LAS-S3) in vitro and in vivo. LAS-S3 showed anti-HSV-1 action with high selectivity indices for HSV-1 KOS (88.1) and AR (189.2), sensitive and resistant to ACV, respectively. LAS-S3 inhibited >80% of HSV-1 infection in different treatment protocols (virucidal, adsorption inhibition, and post-adsorption effects), even at low doses, and showed a preventive effect and DNA and protein synthesis inhibition. The antiherpetic effect was confirmed in vivo by the cosmetic LAS-S3-CRÈME decreasing cutaneous lesions of HSV-1, including the AR strain. LAS-S3 possessed a broad-spectrum mechanism of action acting in the early and post-adsorption stages of HSV-1 infection, and LAS-S3-CRÈME is a potential antiherpetic candidate for patients infected by HSV-1-resistant strains.