Clostridium difficile is the major cause of nosocomial diarrhoea. Several detection methods are available for the laboratory diagnosis of C. difficile, but these vary in terms of sensitivity and specificity. In this study, we compared the performance of three following laboratory tests to detect C. difficile: in-house real-time PCR aiming for toxin B gene (tcdB), EIA for detection of toxins A and B (Premier Toxins A & B) and C. difficile culture in selective medium (bioMerieux). Our results were grouped into three categories as follows: (1) C. difficile-associated diarrhoea (CDAD); (2) asymptomatic carriers; and (3) negative results. Among the 113 patients included in the study, 9 (8.0%) were classified as CDAD, 19 (16.8%) were asymptomatic carriers, 76 (67.2%) had negative results and 9 (8.0%) could not be categorized (positive test for C. difficile toxins only). PCR was found to be the most sensitive diagnostic test in our study, with the potential to be used as a screening method for C. difficile colonization/CDAD. Diagnosis of CDAD would be better performed by a combination of PCR and EIA tests.