Herpes Simplex Virus type 1 (HSV-1) antibodies are found in up to 90 percent of the general population. About 30% of patients who have been exposed to HSV-1 develop recurrent infections, and this degree is continually increasing. In addition, resistance to all major anti-herpetic drugs such as acyclovir (ACV) has been increasingly reported. These observations underscore the importance of discovering new therapeutic tools for the treatment of HSV-1 infections. Bifidobacterium spp. has been studied in various fields including antibacterial and anticancer effect, but the antiviral activity was studied very little. The aim of this study was to test the antiviral activity of Bifidobacterium spp. against HSV-1. The Bifidobacterium adolescentis SPM 0214 used in this study through the screening of 23 Bifidobacterium spp. by plaque assay was assessed the cell viability assay in Vero cells. We also measured the plaque reduction assay and yield reduction assay after B. adolescentis SPM 0214 treatment at concentrations ranging between 10 and 10⁴ μg/mL. The B. adolescentis SPM 0214 was not toxic to Vero cells, and the inhibition of plaque and yield formation was obviously increased compared to those of the control (no additive). Therefore, these results indicate that antiviral activity of B. adolescentis SPM 0214 against HSV-1.