Background: Clostridium difficile-associated disease (CDAD) is the most common infectious antibiotic-associated diarrhea and is a growing health care problem. Prevention of Clostridium difficile infection focuses on clinical and epidemiologic infection control measures. Methods: Between 2008 and 2009, we conducted a retrospective study that showed an incidence of CDAD among the highest reported in the literature. Subsequently, we developed a preventive protocol that was adopted in our hospital in 2010. We then conducted a prospective study to investigate prevalence, incidence, and mortality of CDAD and to compare the results with those of the retrospective study, evaluating adherence to preventive measures and their efficacy. Results: In both studies, prevalence and incidence significantly increased in older patients. Crude prevalence was similar in the 2 studies. The incidence rate increased by 36%, with a significant increase only in the C and D wards. In-hospital mortality rose in both prevalent and incident cases. Regarding adhesion to hospital protocol, 77% of prevalent cases were treated with the required procedure. The highest percentage of isolated patients was achieved in C and D wards. In these wards we detected lower training hours per nurse. However, in 2013, we observed a significant decrease in incidence of CDAD and found a hospital prevalence of 0.33%. Conclusions: Health care personnel education could be more important than the possibility of isolating infected patients in single rooms.