Aim of the study: The aim of this study is to describe the methods of microbiological diagnostic of C. difficile and to investigate the microbiological characteristics of toxigenic strains and demographic characteristics of patients with CDI in the University Hospital of Split from 1 January to 31 December 2020. Materials and methods of the study: Data base of the Department of Clinical Microbiology and Parasitology in the University Hospital of Split was used in this study. All patients for whom a toxigenic strain of C. difficile from a stool sample was detected during this period were included. Microbiological diagnostic of CDI is carried out according to the guidelines of the ESCMID according to the so-called two-stage and three-stage diagnostic procedure. A combined membrane enzyme-linked immunosorbent assay/ immunochromatography test (Savyon Diagnostics Ltd.) was used in this study, which simultaneously tests glutamate dehydrogenase (GDH antigen) and the production of C. difficile toxins A and B. This way, the first and second step of the recommended ESCMID procedure for diagnosing CDI are performed simultaneously. As a confirmatory test, the HG C. difficile molecular test manufactured by HiberGene was used. The test uses loop-mediated isothermal amplification technology to prove the presence of a toxigenic region in the C. difficile genome. Results: In 2020, 1675 stool samples were received at the Department of Clinical Microbiology and Parasitology in the Univeristy Hospital of Split under suspicion of C. difficile infection. Of these, 1265 were negative, and a toxigenic strain of C. difficile was detected in 397 (24 %) samples. Of a total of 397 toxic strains of C. difficile, 3 % were only positive for toxin A production, 5 % of samples were positive for toxin B production, 65 % of samples were positive for toxin A and B production, and 27 % of toxigenic C. difficile strains did not produce toxins at the time of testing. There were 176 men (44 %) and 221 women (56 %). The largest number of patients (66 %) were 65 or older. There were 72 % of hospital patients, and the largest number of them were treated in the Clinic for Internal Medicine (34 %), the Clinic for Infectious Diseases (16 %) and the Clinic for Lung Diseases (14 %). The most common diagnoses are intestinal infections (20 %), other diseases of the digestive system (23 %) and respiratory diseases (16 %).