Interactions between Clostridioides difficile, the gastrointestinal microbiota, and the host mucosal epithelium play important roles in governing the ability of C. difficile to colonize and cause disease. Several in vitro tools have been developed to investigate C. difficile physiology in the presence of microbial communities. In this chapter, we describe a model for studying C. difficile-mucin interactions in the presence of a complex microbiota using continuous flow bioreactors. This model can facilitate mechanistic studies of specific microbes and mucin structures important for C. difficile colonization, complementing findings from animal models. The approach presented here builds upon the preceding chapter’s protocol for generating mucin hydrogels on glass slides and extends it to examine C. difficile adhesion to mucosal surfaces.