The 2006 National Prevalence Survey (ENP) of healthcare-associated infections (HAIs) aimed to provide a snapshot of HAIs in healthcare facilities (HCFs) on a specific day. Conducted in June across all HCFs using a standardized protocol, the survey included inpatients, excluding those admitted that day. The 2,337 participating HCFs encompassed 358,353 patients. The prevalence of infected patients (PPI) was 4.97%, and that of HIs was 5.38%. The most common sites of infection were urinary tract infections (30% of HIs), pneumonia (15%), and surgical site infections (14%). The main microorganisms responsible for HIs were Escherichia coli (25%), Staphylococcus aureus (19%, of which 52% were methicillin-resistant, MRSA), and Pseudomonas aeruginosa (10%, of which 25% were ceftazidime-resistant). The PPI varied according to the type of healthcare facility, length of stay, patient characteristics, and region. When restricted to HIs acquired in 1,351 healthcare facilities participating in both surveys, an analysis adjusted for healthcare facility, department, and patient characteristics shows that between 2001 and 2006, the PPI decreased by 12% and the MRSA PPI by 40%. These results support the positive impact of national plans to combat hospital-acquired infections, particularly for MRSA. (R.A.)