Background: C. difficile infection (CDI) is a common debilitating nosocomial infection associated with high mortality. Several CDI outbreaks have been attributed to ribotypes 027, 017, and 078. Clinical and experimental evidence indicates that the non-pathogenic yeast Saccharomyces boulardii CNCM I-745 (S.b) is effective for the prevention of CDI. However, there is no current evidence suggesting this probiotic can protect from CDI caused by outbreak-associated strains. Methods: We used established hamster models infected with outbreak-associated C. difficile strains to determine whether oral administration of live or heat-inactivated S.b can prevent cecal tissue damage and inflammation. Results: Hamsters infected with C. difficile strain VPI10463 (ribotype 087) and outbreak-associated strains ribotype 017, 027 and 078 developed severe cecal inflammation with mucosal damage, neutrophil infiltration, edema, increased NF-kappaB phosphorylation, and increased pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha) protein expression. Oral gavage of live, but not heated, S.b starting 5 days before C. difficile infection significantly reduced cecal tissue damage, NF-kappaB phosphorylation, and TNFalpha protein expression caused by infection with all strains. Moreover, S.b conditioned medium reduced cell rounding caused by filtered supernatants from all C. difficile strains. S.b conditioned medium also inhibited toxin A- and B-mediated actin cytoskeleton disruption. Conclusions: S.b is effective in preventing C. difficile infection by outbreak-associated via inhibition of the cytotoxic effects of C. difficile toxins.