The discovery and design of antiviral agents have gained unprecedented significance due to the emergence of global health threats. The use of synthetic chemistry has enabled the modification of existing molecules and the creation of entirely novel compounds. In our laboratory, we have enzymatically synthesized a novel bioconjugate, lithocholic acid oleate (LO), derived from lithocholic acid (LCA), a bile acid that has been proven by researchers to exhibit antiviral activity in vitro. The study presented herein describes the preparative synthesis, formulation, and evaluation of LO both in vitro and in vivo for its antiviral activity against human herpes simplex virus 1 (HSV-1) as a model of viral infection. Evaluation of cytotoxicity using A549 cells indicated that a combination of LO (400 μM) and LCA (30 μM) exhibited a favorable safety profile while effectively inhibiting HSV-1 infection comparable to acyclovir treatment. Furthermore, in the in vivo assay, animals treated with an oily formulation containing 7% LO; 0.50% LCA; and 3% oleic acid (OA), 48 h prior to virus exposure, showed results even superior to a 5% acyclovir commercial formulation in terms of scar formation and wound recovery. These promising results enable the development of new preventive products against HSV-1 and probably other viruses.