Our recent Phase 1 trial demonstrated that PC-1005 gel containing 50 μM MIV-150, 14 mM zinc acetate dihydrate and carrageenan (CG) applied daily vaginally for 14 days is safe and well tolerated. Importantly, cervicovaginal lavages (CVLs) collected 4h or 24h after last gel application inhibited HIV-1 and HPV in cell-based assays in a dose-dependent manner (MIV-150 for HIV-1 and CG for HPV). Herein we aimed to determine the anti-HIV and anti-HSV-2 activity of PC-1005 in human cervical explants after in vitro exposure to the gel and to CVLs from participants in the Phase 1 trial. Single HIV-1_BaL infection and HIV-1_BaL/HSV-2 co-infection explant models were utilized. Co-infection with HSV-2 enhanced tissue HIV-1_BaLinfection. In vitro exposure to PC-1005 protected cervical mucosa against HIV-1_BaL (up to 1:300 dilution) in a single challenge and co-challenge models. CG gel (PC-525) provided some barrier effect against HIV-1_BaL at the 1:100 dilution in a single challenge model, but not in the co-challenge model. Both PC-1005 and PC-525 at the 1:100 dilution inhibited HSV-2 infection, pointing to a CG-mediated protection. MIV-150 and CG in CVLs inhibited HIV (single challenge or co-challenge models) and HSV-2 infections in explants in a dose-dependent manner (p< 0.05). Stronger inhibition of HIV-1 infection by CVLs collected 4h post last gel administration was observed when compared to infection detected in the presence of baseline CVLs. The anti-HIV and anti-HSV-2 activity of PC-1005 gel in vitro and CVLs in human ectocervical explants supports the further development of PC-1005 gel as a broad-spectrum on-demand microbicide.