The present study examined the efficacy of using multiple mechanisms as part of a topical microbicide to inactivate herpes simplex virus (HSV) by combining theaflavin-3,3'-digallate (TF-3) and lactic acid (LA) over the pH range of 4.0 to 5.7 to mimic conditions in the female reproductive tract. Six clinical isolates of HSV-2 and two clinical isolates of HSV-1 were almost completely inactivated when TF-3 (100 μM) was present with LA over the pH range of 4.5 to 5.7, whereas four additional HSV-1 clinical isolates required TF-3 concentrations of 250 to 500 μM for comparable virus titer reduction. LA (1%) alone at pH 4.0 reduced the titers of laboratory and clinical isolates of HSV-1 and HSV-2 by ≥ 5 log10, but most LA-dependent antiviral activity was lost at a pH of ≥ 4.5. When HSV-1 and HSV-2 were incubated at pH 4.0 without LA virus titers were not reduced. At pH 4.0, HSV-1 and HSV-2 titers were reduced 5 log(10) in 20 min by LA alone. TF-3 reduced HSV-2 titers by 5 log10 in 20 to 30 min at pH 4.5, whereas HSV-1 required 60 min for comparable inactivation. Mixtures of TF-3 and LA stored at 37 °C for 1 month at pH 4.0 to 5.7 maintained antiviral activity. Semen, but not cervical vaginal fluid, decreased LA-dependent antiviral activity at pH 4.0, but adding TF-3 to the mixture reduced HSV titers by 4 to 5 log10. These results indicate that a combination microbicide containing TF-3 and LA could reduce HSV transmission.