Clostridium difficileinfection (CDI) is a major healthcare-associated disease worldwide. Recurring infections and increasing antibiotic resistance have complicated treatment of CDI. WhileC. difficilespores are important for transmission and persistence of CDI, other factors such as gut colonization and formation of bacterial communities in the gut may also contribute to pathogenesis and persistence, but have not been well investigated. Recently, we reported that important clinicalC. difficilestrains are able to form composite biofilms in vitro.C. difficilebiofilm formation is a complex process, modulated by several different factors, including cell surface components and regulators. We also reported that bacteria within biofilms are more resistant to high concentrations of vancomycin, the antibiotic of choice for treatment of CDI. Here we summarize our recent findings and discuss the implications of biofilm formation by this anaerobic gut pathogen in disease pathogenesis and treatment.