Lactobacillus probiotics have been proposed as an antibiotic-sparing prevention strategy for urinary tract infections (UTIs). Our objective was to examine the relative ability of the 4 most common vaginal Lactobacillus species to inhibit the growth of Escherichia coli, the most common cause of UTIs. Conditioned media (CM) was created from 4 laboratory strains of Lactobacillus species: Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, and Lactobacillus iners, and 6 clinical strains of L. crispatus. One laboratory strain of E. coli was cocultured with each CM, as well as with various acidic solutions. Three clinical strains of E. coli from women with acute cystitis were cocultured with the CM from a laboratory strain of L. crispatus. Bacterial growth was compared between experimental variants and media control using analysis of variance. Growth of E. coli was inhibited by CM from L. crispatus, L. jensenii, and L. gasseri, but not L. iners. The magnitude of inhibition was correlated with the pH of the individual CM and the concentration of D-lactic acid. Different acids inhibited E. coli growth in proportion to the pH of the acid solution. Similar levels of inhibition were seen when L. crispatus was incubated with clinical E. coli strains as with laboratory E. coli. Three of the most common vaginal Lactobacillus species inhibit E. coli growth, likely through creating a low pH environment. However, L. iners, one of the most common species found after menopause, does not. These findings might be leveraged to more effectively manage UTIs.