Clostridioides difficile (syn. Clostridium difficile) is a bacterium known for causing serious diarrheal infections, and may also cause colon cancer. It is known also as C. difficile, or C. diff (/siː dɪf/), and is a Gram-positive species of spore-forming bacteria. Clostridioides spp. are anaerobic, motile bacteria, ubiquitous in nature and especially prevalent in soil. Its vegetative cells are rod-shaped, pleomorphic, and occur in pairs or short chains. Under the microscope, they appear as long, irregular (often drumstick- or spindle-shaped) cells with a bulge at their terminal ends (forms subterminal spores). C. difficile cells show optimum growth on blood agar at human body temperatures in the absence of oxygen. C. difficile is catalase- and superoxide dismutase-negative, and produces up to three types of toxins: enterotoxin A, cytotoxin B and Clostridioides difficile transferase. Under stress conditions, the bacteria produce spores that tolerate extreme conditions that the active bacteria cannot tolerate.

Clostridioides difficile is an important human pathogen; according to the CDC, in 2017 there were 223,900 cases in hospitalized patients and 12,800 deaths in the United States. Although C. difficile is known as a hospital- and antibiotic-associated pathogen, at most one third of infections can be traced to transmission from an infected person in hospitals, and only a small number of antibiotics are directly associated with an elevated risk of developing a C. difficile infection (CDI), namely vancomycin, clindamycin, fluoroquinolones and cephalosporins. Most infections are acquired outside of hospitals, and most antibiotics have similar elevated risk of infection on par with many non-antibiotic risk factors, such as using stool softeners and receiving an enema.

Clostridioides difficile can become established in the human colon without causing disease. Although early estimates indicated that C. difficile was present in 2–5% of the adult population, later research indicated that colonization is closely associated with a history of unrelated diarrheal illnesses, such as food poisoning or laxative abuse. Individuals with no history of gastrointestinal disturbances appear unlikely to become asymptomatic carriers. These carriers are thought to be a major infection reservoir.

The species was transferred from the genus Clostridium to Clostridioides in 2016, thus giving it the binomial Clostridioides difficile. This new name reflects the taxonomic differences between this species and members of the genus Clostridium, while maintaining the common name as C. difficile . As of 2018^[update], the only other species in this new genus is Clostridioides mangenotii (formerly known as Clostridium mangenotii).

Pathogenic C. difficile strains produce multiple toxins. The best-characterized are enterotoxin (C. difficile toxin A) and cytotoxin (C. difficile toxin B), both of which may produce diarrhea and inflammation in infected patients (C. difficile colitis), although their relative contributions have been debated. The diarrhea may range from a few days of intestinal fluid loss to life-threatening pseudomembranous colitis, which is associated with intense colon inflammation and pseudomembrane formation on the intestinal mucosal surface. This may progress to toxic megacolon, a severe form of colonic distention that can put a patient at risk for colon perforation, sepsis and shock. Toxins A and B are glucosyltransferases that target and inactivate the Rho family of GTPases. Toxin B (cytotoxin) induces actin depolymerization by a mechanism correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins. A binary toxin (AB toxin), but its role in disease is not fully understood.

Additional virulence factors include an adhesion factor that mediates the binding to human colonic cells and a hyaluronidase. The bacterium also produces the chemical para-cresol, which inhibits the growth of other microbes in its vicinity and allows it to outcompete normal human gut flora.

Antibiotic treatment of C. difficile infections may be difficult, due both to antibiotic resistance and physiological factors of the bacterium (spore formation, protective effects of the pseudomembrane). A highly toxic strain of C. difficile, resistant to fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, said to be causing geographically dispersed outbreaks in North America, was reported in 2005. In 2005 the US Centers for Disease Control in Atlanta warned of an epidemic strain with increased virulence, antibiotic resistance, or both. In 2018, resistance to other antibiotics such as metronidazole, the first choice of antimicrobial drug when treating CDI, was observed in up to 12% of clinical isolates. While treatment with various antibiotics continues, more diverse and stronger resistance will continue in C. difficile populations.

Clostridioides difficile spores resist many disinfectants, including high concentrations of bleach.