Topical microbicides are considered an affordable choice for the prevention of sexually transmitted diseases in women. We have developed a comprehensive testing program for preclinical microbicide development. Over the years, we have tested several thousand compounds for use as topical microbicides in a series of cell-based assays addressing HIV-1 efficacy and toxicity. Recently, we compared historical data of the spermicide nonoxynol-9 (N-9) in a multi-center study and found that the HIV-1 efficacy paralleled its toxicity. Intra-assay, inter-assay, and inter-laboratory variability for toxicity were remarkably consistent. In a recent clinical trial, N-9 was found to enhance HIV-1 infection, thus confirming the preclinical toxicity data. In addition to N-9, lemon and lime juices have been proposed and used as contraceptives and were recently shown to exhibit in vitro activity against HIV-1. Therefore, we tested freshly prepared lemon juice, lime juice, and household vinegar (concentration = 100%) for HIV-1 efficacy and toxicity and for effect on beneficial Lactobacillus species. In all assays, the therapeutic index was <10, due to toxicity of the juices and vinegar to cells (mean TC50 of lemon juice = 5.6%, mean TC50 of lime juice = 4.9%, and mean TC50 of vinegar 0.1%). Ten percent lemon or lime juice were not toxic to beneficial Lactobacillus species, in contrast to 10% vinegar which was highly toxic. Our preclinical data indicate that candidate topical microbicides should be moved forward into clinical trials with caution.