Mucosal viruses interact with the host microbiome before infecting and replicating with the host. These interactions can influence infectivity, replication and transmission, but have primarily been studied in the mammalian gut mucosa. The role of these interactions, if any, in the vaginal mucosa remains unclear. Here we show that metabolically inactive lactobacilli, including the dominant vaginal species Lactobacillus crispatus, reduce HSV-2 infection in vitro, suggesting a role for the bacterial cell body in blocking HSV-2 infection. Using cell culture and a mouse model of intravaginal HSV-2 infection, we demonstrate that this protection is provided by the presence of lactobacillus cell wall. Robust protection from HSV-2 requires peptidoglycan (PG) at time of infection and correlates with reduced infectious virus in the vagina early in infection. Using commercially available PG, we demonstrate that other gram-positive PG protects against HSV-2 and HSV-1. We also show that PG protection against HSV-2 depends on long PG chains, suggesting that PG acts as a physical barrier to viral entry. Collectively, this work suggests a role for lactobacilli cell wall in blocking HSV infection independently of the immune system, providing a potential mechanism by which lactobacilli reduce HSV in humans.