Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 pre-exposure followed by co-challenge with HSV-2 and simian/HIV (SHIV-RT). Using this model, we showed that a gel containing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal co-challenge with SHIV-RT and HSV-2 in HSV-2-naïve macaques. All animals (6/6) became HSV-2-infected, with 4 of 6 co-infected with SHIV-RT. In a control group co-challenged with SHIV-RT and UV-inactivated HSV-2, 2 of 4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naïve animals full protection from SHIV-RT for at least 8 hours (h), MZC (vs. CG) was applied daily for 14 days (d) followed by co-challenge 8h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque co-infection model to evaluate broad-spectrum microbicides.