Hydrogen peroxide production by vaginal lactobacilli represents one of the most important defense mechanisms against vaginal colonization by undesirable microorganisms. To quantify the ability of a collection of 45 vaginal Lactobacillus strains to generate H2O2, we first compared three published colorimetric methods. It was found that the use of DA-64 as a substrate rendered the highest sensitivity, while tetramethyl-benzidine (TMB) maintained its linearity from nanomolar to millimolar H2O2 concentrations. Generation of H2O2 was found to be especially common and strong for L. jensenii strains, while it was variable among L. crispatus and L. gasseri strains. Biosynthesis of H2O2 only occurred upon agitation of the cultures, but the H2O2-producing machinery was already present in them before aeration started. Calcium, magnesium, manganese, and zinc ions did not affect H2O2 production, while Cu2+ inhibited the growth of Lactobacillus jensenii CECT 4306, which was chosen as a model strain. Cultures with Fe3+, hemin, and hemoglobin did not accumulate H2O2, Fe3+ activated an extracellular peroxidase that destroyed the H2O2 being produced by the cultures. This protected the lactobacilli against its antimicrobial effect. The production of the enzyme appears to be constitutive, the Fe3+ ions being a necessary cofactor of the reaction.