Aims In this study, we evaluated the antiviral activity of subtilosin, a cyclical peptide isolated from Bacillus amyloliquefaciens, against herpes simplex virus type 2 (HSV‐2) in cell cultures and we investigated subtilosin mode of action. Methods and Results We determined, using a virus yield inhibition assay, that noncytotoxic concentrations of subtilosin inhibit HSV‐2 replication in Vero cell cultures. Subtilosin strongly inhibited extracellular and total virus production even when it was added at 8 h postinfection indicating that not only virus release but also viral particle formation is impeded by the antiviral peptide. Although viral glycoprotein gD level of expression is not affected by the bacteriocin, an altered pattern of gD intracellular localization was detected by immunofluorescence assay in subtilosin‐treated culture. On the other hand, at high concentrations, subtilosin displays virucidal action. Conclusions Subtilosin displays antiviral and virucidal actions against HSV‐2. The target of subtilosin inhibitory effect would be late stages of the viral replicative cycle such as viral glycoprotein intracellular transport. Significance and Impact of the Study Given its antimicrobial activity and its safety for human tissues, subtilosin could represent a valuable alternative to be considered in the development of new microbicide formulations.