The antimicrobial and anti-adhesive effects of extracellular factors from 27 strains of bifidobacteria isolated from healthy infants were tested against two reference strains of Clostridium difficile (ATCC 9689 and ATCC 43593). All bifidobacterial supernatants at pHs between 5.0 and 4.1 were able to produce strain-dependent growth inhibition of clostridia in the agar-diffusion assay. Six strains of Bifidobacterium produced during growth extracellular factors able to antagonize the adhesion of C. difficile ATCC 9689 and ATCC 43593 to cultured human enterocytes (Caco-2/TC7). Factors responsible for the anti-adhesive effect were thermolabile, active at neutral pH and unaffected by proteolytic cleavage (proteinase K and chymotrypsin). Results of the present paper show the potential of selected bifidobacteria to antagonize key mechanisms involved in the virulence of C. difficile.