Clostridioides difficile is a Gram-positive, spore-forming anaerobic enteropathogen responsible for a wide spectrum of clinical diseases ranging from mild diarrhoea to pseudomembranous colitis. It is the first cause of healthcare-associated diarrhoeas, but community-associated Clostridioides difficile infections (CDI) are increasingly reported in patients without the common risk factors (age > 65 years, previous antibiotic treatment). The main C. difficile virulence factors are toxins A (TcdA) and B (TcdB), and in some cases the binary toxin. The CDI incidence has increased in Europe since the early 2000s, then decreased to reach approximately 4 cases/10,000 patients/days. C. difficile should be tested only in diarrheal stools. Children less than 3 years old are frequently colonized, therefore CDI diagnosis should be carried out only in specific cases (outbreak, Hirschsprung disease). No stand-alone method can be used for the CDI diagnosis. The European Society for Clinical Microbiology and Infectious Diseases (ESCMID) recommends a two-step algorithm with a sensitive screening test (molecular assay or glutamate dehydrogenase immunochromatographic assay). If the screening test is negative, the CDI diagnosis can be ruled out. If the screening test is positive, a second highly specific test should be used, such as toxin A/B immunochromatographic assay.