Limited information is available on the burden and epidemiology of Clostridium difficile infection (CDI) in Spain. The present report communicates the secular trends in prevalence of CDI among hospitalised patients in Spain from 1999 through 2007. Data were obtained through the EPINE study (Estudio de prevalencia de las infecciones nosocomiales en los hospitales españoles), a point prevalence study series of nosocomial infections among patients admitted to hospital in Spain. A total of 378 cases with CDI were identified. Median age was 74 years. Prevalence rates of CDI increased from 3.9 to 12.2 cases per 10,000 hospitalised patients and showed a significantly increasing secular trend from 1999 through 2007 (prevalence rate ratio per each year increment 1.09; 95% CI 1.05 - 1.14). Percentage of hospitalised patients receiving antimicrobials increased linearly from 36.0% in 1999 to 40.7% in 2007 (p <0.001) and was strongly correlated to CDI prevalence (R square = 0.73; regression coefficient =1.194, 95% CI= 1.192 - 1.196).