This study presents a series of experiments carried out in order to elucidate the role of H2O2 in antimicrobial activity of lactobacilli. Vaginal swabs were collected from 60 premenopausal women and checked for pH and Nugent score, and Lactobacillus species were cultured, phenotyped and genotyped. The main outcome measures involved: (1) species of vaginal lactobacilli most effective in liberating H2O2, (2) minimal microbicidal concentrations of added H2O2, (3) kinetics of H2O2 liberation in relation to oxygen tension, (4) antimicrobial activity of pure H2O2 versus one produced by selected vaginal lactobacilli and the total activity of their culture supernatants. Results showed that H2O2 was liberated especially by: Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus johnsonii and L. gasseri. Hydrogen peroxide reached concentrations from 0.05 to 1.0 mM, which under intensive aeration increased even up to 1.8 mM. Microorganisms related to vaginal pathologies show varied resistance to the action of pure H2O2. Most potent inhibitory activity against bacteria and yeasts was presented by Lactobacillus culture supernate producing H2O2, followed by the nonproducing strain and pure H2O2. To conclude - the antimicrobial activity of lactobacilli is a summation of various inhibitory mechanisms in which H2O2 plays some but not a crucial role, in addition to other substances.