Nosocomial Clostridium difficile-associated disease (CDAD) is a common infection in hospitals. A matched case-control study was carried out to determine hospital-wide excess costs due to CDAD. Cases were assessed by prospective hospital-wide surveillance in a tertiary care university hospital in 2006. Nosocomial cases of CDAD (>72h after admission) were matched to control patients without CDAD in a ratio 1:3 using the same diagnosis-related group in the same year, for a hospital stay at least as long as the time of risk of the CDAD cases before infection and a Charlson comorbidity index +/-1. Data on overall costs per case were provided by the finance department. Matching was possible for 45 nosocomial CDAD cases. The difference in the length of stay showed that CDAD cases stayed significantly longer (median 7 days; P=0.006) than their matched controls. The average cost per CDAD patient was euro 33,840. The difference in the cost per patient showed that the cost for CDAD patients was significantly more than for their matched controls (median euro 7,147; 95% confidence interval: 4,067-9,276). Nosocomial CDAD is associated with high costs for healthcare systems. Clinicians should be aware of the financial impact of this disease and the application of appropriate infection control measures is recommended to reduce spread.