Clostridium (Clostridioides) difficile infections (CDIs) are among the most frequent healthcareassociated infections in Serbia. In 2013, Serbia participated in the European Clostridium difficile Infection Surveillance Network (ECDIS-Net) who launched a pilot study to enhance laboratory capacity and standardize surveillance for CDI. Two clinics of Clinical Center of Serbia [Clinic for Infectious and Tropical Diseases (CITD) and Clinic of Orthopedic Surgery and Traumatology (COT)] from Belgrade and one general hospital from another metropolitan area of Serbia, Užice, participated. During a period of 3 months in 2013, all patients with diagnosed CDI were included. The CDI incidence rates in CITD, COT, and General Hospital Užice were 19.0, 12.2, and 3.9 per 10,000 patient-days, respectively. In total, 49 patients were enrolled in the study with average age of 72 years. A complicated course of CDI was found in 14.3% of all patients. Six (12.2%) of 49 patients died, but not attributable to CDI. Of 39 C. difficile isolates, available for ribotyping, 78.9% belonged to ribotype 027; other PCR ribotypes were 001, 015, 002, 005, 010, 014, and 276. Antimicrobial susceptibility testing revealed low levels of MIC 50 and MIC 90 for metronidazole (0.5 μg/ml both) and vancomycin (0.25 and 0.5 μg/ml), while 28 strains of ribotype 027 were resistant to moxifloxacin with MIC ≥4 μg/ml. National surveillance is important to obtain more insight in the epidemiology of CDI and to compare the results with other European countries. This study by ECDIS-Net gives bases for a national surveillance of CDI in Serbia.