We estimated the direct additional medical costs of nosocomial infections (NI) using a cohort study in acute and longer-term care at Nîmes University Hospital in France. Patients hospitalised between May 2001 and January 2003 with NI were considered as exposed; all others were eligible as non-exposed. Thirty patients were randomly chosen for each site of infection: respiratory tract, bloodstream, surgical site, urinary tract and other sites for a total of 150 exposed patients. Each exposed patient was matched with a non-exposed patient according to gender, age, severity of the underlying disease, diagnosis according to hospital discharge records, ward type and length of hospitalisation before inclusion. Additional direct medical costs for the exposed patients compared to the non-exposed and the difference between actual costs and the diagnosis-related group rate were measured. Costs resulting from laboratory tests, radiology, surgery and exploratory examinations, and antimicrobial agents were estimated to be Euro2421 for a respiratory tract infection, Euro1814 for a surgical site infection, Euro953 for a bloodstream infection and Euro574 for a urinary tract infection. Total additional costs of NI (direct medical costs and costs of extra length of stay) in acute care were estimated to be up to Euro3.2 million per year (95% confidence interval: 2,275,063-4,132,157). In conclusion, both prevention of avoidable NI and better estimation of the actual costs of NI should be priorities for all healthcare facilities.