Pharmaceutical excipients are widely used in vaginal drug products. The epithelial integrity of the cervicovaginal tissue is important for HIV-1 prevention. However, the effect of excipients on cervicovaginal epithelium remains unknown. This study aims to assess the effect of vaginal product excipients on the integrity of human cervicovaginal epithelium and their effect on a lead HIV prevention candidate tenofovir. In the current study, nine excipients commonly used in vaginal formulations were incubated for 6 hours with excised human ectocervical epithelia. The effect of excipients on epithelial integrity was examined by measuring the transepithelial electrical resistance (TEER), epithelial morphology, paracellular/transcellular permeability, cell viability, and the efficacy of tenofovir for preventing HIV-1 infection to ex vivo cultured ectocervix. We found that disodium EDTA, sorbic acid, and benzoic acid had no effect on the tissue TEER. Butylated hydroxyanisole, glycerin, propylene glycol, methylparaben, and propylparaben slightly-to-moderately decreased tissue TEER, while citric acid significantly decreased the TEER in a time-dependent manner. Also, tissue morphology observed post-exposure correlated well with TEER data, however, there is less correlations between paracellular permeability and TEER data after exposure to different excipients. In addition, treatment with EDTA, methylparaben and propylene glycol at tested levels had no effect on the efficacy of tenofovir in preventing tissue HIV-1 infection. In conclusion, the combined measurements of TEER, morphology, permeability, and viability using human cervicovaginal tissue represent a clinically relevant platform for safety evaluation of excipients and formulated products for HIV-1 prevention.