AIMS: This study aims to evaluate the antiviral activity of sophorolipids against herpes simplex virus type 1 (HSV-1) and develop an anti-herpetic formulation for treatment of cutaneous lesions caused by HSV-1. METHODS AND RESULTS: The antiherpetic activity of sophorolipids was evaluated in vitro against both sensitive (KOS) and acyclovir-resistant HSV-1 strains. used as a model to assess the antiviral activity of sophorolipids against non-enveloped viruses Poliovirus was used as a model to assess the antiviral activity of sophorolipids against non-enveloped viruses. The results showed that sophorolipids exhibit effective antiviral activity against both strains with low cytotoxicity to VERO cells. However, antiviral activity against poliovirus was not observed, suggesting that sophorolipids specifically target enveloped virus. In vivo, the sophorolipid-based cream formulation demonstrated good stability and efficacy in reducing herpetic lesions, including those caused by the drug-resistant strain. Promising antiviral activity was confirmed through histopathological analysis, indicating a reduced occurrence of tissue damage in the treated group compared to the viral control. CONCLUSIONS: Sophorolipids, whether isolated or incorporated as an active ingredient in a cream formulation, represent a promising and innovative alternative for the treatment of cutaneous lesions caused by HSV-1, including strains resistant to the reference drug.