Toxigenic Clostridium difficile is responsible for antibiotic-associated diarrhoea and other diseases. The increasing frequency and severity is attributed to highly-virulent ribotypes such as 027. The aim of the study was to collect epidemiological and molecular data for C. difficile isolates during 2009-2013 in the Central Hospital of Bolzano, Northern Italy. Stool samples from inpatients of the Bolzano Central Hospital were screened for toxins A and B, and C. difficile was cultured and tested for antibiotic susceptibility. PCRs were performed for genes of toxin A, toxin B, binary toxin and ribotyping. During the period 2009-13 from 320 patients (9% of patients tested) at least one stool sample proved positive for C. difficile toxins, and incidences for all hospital inpatients per 10,000 patient days (per 1,000 admissions) varied between 2.2 (1.5) and 4.3 (3.0). Out of 138 isolates (43% of total isolates were studied), 24 different ribotypes were identified. Isolates with ribotype 027 were predominant (38%), followed by 018 (13%) and 607 (10%). Whereas for ribotype 018 a significant decrease was seen during the five-year period, ribotype 027 increased significantly from 0% in 2009 to 64% in 2012, decreasing then to 10% in 2013. Isolates were sensitive to metronidazole and vancomycin, whereas isolates of the three major ribotypes were resistant to moxifloxacin. Our data indicates a significant change in C. difficile incidence rates and ribotype frequencies during the five-year period in the Central Hospital in Bolzano.