Background: Clostridium difficile (C. difficile), a Gram-positive spore-forming bacillus, is the most common etiologic agent of antibiotic-associated diarrhea. The clinical presentation of Clostridium difficile-associated disease (CDAD) occurs secondary to the production of two exotoxins; toxin A, an enterotoxin and toxin B, a cytotoxin. Aim: to assess the frequency of C. difficile infection in hospitalized children in Suez Canal University Hospital for the purpose of prevention and control. Patients and Methods: This study was conducted as a cross-sectional study to detect C. difficile infection in 90 hospitalized children in Pediatric inpatient ward in Suez Canal University Hospital selected through a simple random sample. History taking as well as complete stool analysis and enzyme immunoassay to determine toxins A and B of C. difficile in stool samples were done. Results: Determination of toxins A and B of C. difficile in stool samples was found positive in 8.9% of the studied sample. Prevalence of C. difficile was slightly higher in infants than in children and in males more than in females. All 8 patients (100%) with Clostridium difficile infection (CDI) presented by diarrhea, 25% of them presented by abdominal pain and none of them presented by bloody stool. C. difficile infection was highest among patients having Cephalosporin (18.2%) followed by combination therapy between penicillin and cephalosporin (8.6%) while the lowest prevalence was among those on Penicillins only (4.8%). Conclusion: C. difficile infection is prevalent in hospitalized children receiving antibiotics therapy causing a wide variety of GIT symptoms mainly diarrhea.