Breast-fed children, compared with the bottle-fed ones, have a lower incidence of acute gastroenteritis due to the presence of several antiinfective factors in human milk. The aim of this work is to study the ability of human milk oligosaccharides to prevent infections related to some common pathogenic bacteria. Oligosaccharides of human milk were fractionated by gel-filtration and characterized by thin-layer chromatography and high-performance anion exchange chromatography. Fractions obtained contained, respectively, 1) acidic oligosaccharides, 2) neutral high-molecular-weight oligosaccharides, and 3) neutral low-molecular-weight oligosaccharides. Experiments were carried out to study the ability of oligosaccharides in inhibiting the adhesion of three intestinal microorganisms (enteropathogenic Escherichia coli serotype O119, Vibrio cholerae, and Salmonella fyris) to differentiated Caco-2 cells. The study showed that the acidic fraction had an antiadhesive effect on the all three pathogenic strains studied (with different degrees of inhibition). The neutral high-molecular-weight fraction significantly inhibited the adhesion of E. coli O119 and V. cholerae, but not that of S. fyris; the neutral low-molecular-weight fraction was effective toward E. coli O119 and S. fyris but not V. cholerae. Our results demonstrate that human milk oligosaccharides inhibit the adhesion to epithelial cells not only of common pathogens like E. coli but also for the first time of other aggressive bacteria as V. cholerae and S. fyris. Consequently, oligosaccharides are one of the important defensive factors contained in human milk against acute diarrheal infections of breast-fed infants.