Over-the-counter personal lubricants are used frequently during vaginal and anal intercourse, but they have not been extensively tested for biological effects that might influence HIV transmission. We evaluated the in vitro toxicity anti-HIV-1 activity and osmolality of popular lubricants. A total of 41 lubricants were examined and compared to Gynol II and Carraguard as positive and negative controls for toxicity, respectively. Cytotoxicity was assessed using the XTT assay. The MAGI assay with R5 and X4 HIV-1 laboratory strains was used to evaluate antiviral activity. The effect of the lubricants on differentiated Caco-2 cell monolayers (transepithelial electrical resistance, TEER) was also measured. None of the lubricants tested showed significant activity against HIV-1. Surprisingly, four of them, Astroglide Liquid, Astroglide Warming Liquid, Astroglide Glycerin & Paraben-Free Liquid, and Astroglide Silken Secret, significantly enhanced HIV-1 replication (p < 0.0001). A common ingredient in three of these preparations is polyquaternium-15. In vitro testing of a chemically related compound (MADQUAT) confirmed that this similarly augmented HIV-1 replication. Most of the lubricants were found to be hyperosmolar and the TEER value dropped approximately 60% 2 h after exposure to all lubricants tested. Cells treated with Carraguard, saline, and cell controls maintained about 100% initial TEER value after 2–6 h. We have identified four lubricants that significantly increase HIV-1 replication in vitro. In addition, the epithelial damage caused by these and many other lubricants may have implications for enhancing HIV transmission in vivo. These data emphasize the importance of performing more rigorous safety testing on these products.