The obligate anaerobe Clostridium difficile is the leading cause of healthcareassociated intestinal infections and is increasingly involved in diarrhea in the community. The contaminating forms of C. difficile are the resistant spores. In case of compromised gut microbiota (dysbiosis), spores (endogenous or spores acquired from environment) are able to germinate and the outgrowing cells may multiply, leading to colonization of the host. Colonization by toxigenic strains can either remain asymptomatic (carriage) or lead to C. difficile infection (CDI). Symptoms of CDI range from mild diarrhea to severe pseudomembranous colitis. In addition, about 25% of patients experience CDI recurrence, either as reinfection or relapse. Relapse is caused by the initial strain that has persisted despite specific antibiotic treatment. The features of intestinal colonization by C. difficile, whether initial asymptomatic carriage or persitence leading to relapse, are not fully understood, particularly the way in which C. difficile associates to the mucosa. This could occur in the form of spores or of vegetative cells that may be embedded or not in a biofilm. Indeed, multiple relapses are suggestive of chronic infection, which raises the possibility of C. difficile biofilm formation in vivo. Data on C. difficile attachment to human gut tissue are scarce but, during acute infection, C. difficile does not seem to be associated in a biofilm structure. Other data come from studies in various animal models. In conventional dysbiotic mice, C. difficile has been found associated with bacterial communities within the outer mucus layer that could evoke a pluri-species mucosal biofilm. In our lab, we developped a monoxenic mouse model in which several strains of C. difficile are able to form micro-colonies embedded in a polysaccharidic matrix adherent to the mucus. The possible organization of C. difficile in biofilm within the gut will be discussed considering the pathophysiology of CDI and the possible inducers of C. difficile biofilm formation in vivo.