Clostridioides difficile infection (CDI), previously Clostridium difficile infection, is a symptomatic infection of the large intestine caused by the spore-forming anaerobic, gram-positive bacterium Clostridioides difficile. CDI is an important healthcare-associated disease worldwide, characterized by high levels of recurrence, morbidity, and mortality. CDI is observed at a higher rate in immunocompromised patients after antimicrobial therapy, with antibiotics disrupting the commensal microbiota and promoting C. difficile colonization of the gastrointestinal tract.A rise in clinical isolates resistant to multiple antibiotics and the reduced susceptibility to the most commonly used antibiotic molecules have made the treatment of CDI more complicated, allowing the persistence of C. difficile in the intestinal environment.Gut colonization and biofilm formation have been suggested to contribute to the pathogenesis and persistence of C. difficile. In fact, biofilm growth is considered as a serious threat because of the related antimicrobial tolerance that makes antibiotic therapy often ineffective. This is the reason why the involvement of C. difficile biofilm in the pathogenesis and recurrence of CDI is attracting more and more interest, and the mechanisms underlying biofilm formation of C. difficile as well as the role of biofilm in CDI are increasingly being studied by researchers in the field.Findings on C. difficile biofilm, possible implications in CDI pathogenesis and treatment, efficacy of currently available antibiotics in treating biofilm-forming C. difficile strains, and some antimicrobial alternatives under investigation will be discussed here.