Purpose: Toxins produced by Clostridioides difficile infection (CDI) can cause enteritis and diarrhea. Although the number of pediatric CDI cases is increasing, the clinical management of pediatric CDI, including patient characteristics and prognosis, remains unclear. This study aimed to elucidate the background and clinical course of patients with CDI and evaluate the reliability of diagnostic tests in a tertiary pediatric hospital in Japan. Methods: We retrospectively analyzed the clinical data of children diagnosed with CDI between 2011 and 2021 at the Saitama Children’s Medical Center in Saitama, Japan. Results: During the study period, 1,252 C. difficile antigen/toxin tests were performed, and 37 patients were diagnosed with CDI. The main underlying diseases among the patients were hematological and malignant disorders and gastrointestinal diseases, including inflammatory bowel disease (IBD) (59.4%). Two patients (5.4%) had an unremarkable medical history. Among the 37 patients, 27 (73.0%) were immunocompromised, 25 (67.6%) had a history of antibiotic use within the past two months, and 6 (16.2%) were negative on the initial test but were positive on the second test. Finally, 28 patients (75.7%) required primary antibiotic therapy only, and two patients with IBD required additional antibiotic therapy as secondary treatment. Conclusion The number of pediatric patients with CDI is increasing. Both a comprehensive interview, including underlying diseases and history of antibiotic use, and an understanding of the features of clinical examinations should be emphasized to appropriately diagnose and treat CDI.