The obligate anaerobe Clostridium difficile is the leading cause of healthcare-associated diarrhea and has also been involved in antibiotic-associated diarrhea in the community. In patients with compromised gut microbiota (dysbiosis) due to antibiotic usage and/or underlying diseases, spores are able to germinate and the outgrowing cells may persist and multiply, leading to the colonization process. Colonization by toxigenic strains can either remain asymptomatic (carriage) or lead to C. difficile infection (CDI), with symptoms ranging from mild diarrhea to fulminant 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. Multiple relapses are suggestive of chronic infection, which raises the possibility of C. difficile biofilm formation in vivo. The features of gut colonization by C. difficile are not fully understood, particularly those concerning the way in which C. difficile associates to the mucosa. This could occur in the form of spores or of vegetative cells, embedded or not in a biofilm. Data on C. difficile attachment to human gut tissue are scarce, and most of the information comes from studies in animal models. Several methods have been used to gain information on how C. difficile associates to the mucosa. This overview will present the different methods used, the type of information they can provide and the main results obtained. In particular, C. difficile has been found associated with bacterial communities within the outer mucus layer in dysbiotic mice or as micro-colonies embedded in a polysaccharidic matrix adherent to the mucus in a monomicrobial mouse model. The possible organization of C. difficile in biofilm within the gut will be discussed considering the pathophysiology of CDI.