The vaginal microenvironment is key in mediating susceptibility to sexually transmitted infections. A polymicrobial environment with reduced Lactobacilllus spp. is characteristic of vaginal dysbiosis, associated with increased production of several short chain fatty acids (SCFAs), vaginal inflammation and an increased risk of HIV-1 acquisition. In contrast, a eubiotic vaginal microbiome (VMB), dominated by Lactobacillus spp. correlates with increased production of lactic acid (LA), an acidic milieu and protection against HIV-1. Vaginal metabolites, specifically LA and SCFAs including butyric, succinic and acetic acids are associated with modulation of HIV-1 risk. We assessed the impact of combined and individual SCFAs and LA on vaginal epithelial cells (VK2) grown in air-liquid interface cultures. Treatment of VK2 cells with eubiotic SCFA + LA mixture showed increased epithelial barrier integrity, reduced FITC dextran leakage and enhanced expression of cell-cell adhesion proteins. Treatment with dysbiotic SCFA + LA mixture diminished epithelial barrier integrity, increased NFκB activation and inflammatory mediators: TNF-α, IL-6, IL-8 and RANTES. LA was found to be the primary contributor of the beneficial effects. Eubiotic SCFA + LA mixture ameliorated HIV-1 mediated barrier disruption and HIV-1 leakage, whereas dysbiotic SCFA + LA treatment exacerbated HIV-1 effects. These findings indicate a key role for LA in future prophylactic strategies.