The National Prevalence Survey (ENP) on healthcare-associated infections (HAIs) in healthcare facilities is conducted every five years. In 2017, its objective was to measure the prevalence of these infections and to describe the patients infected in healthcare facilities. The survey was conducted on a representative sample of French healthcare facilities using random sampling stratified by region and facility category. Between May 15 and June 30, 2017, 403 healthcare facilities participated, including 80,988 patients. In 2017, one in 20 patients hospitalized in healthcare facilities was infected (4.98%, 95% CI: [4.62–5.36]). This prevalence did not decrease between 2012 and 2017, whereas previous surveys had shown a steady decline since 2001. In 2017, the prevalence of infected patients increased among short-stay patients, particularly in surgical units, compared to 2012. It also increased among patients with certain risk factors for infection: having a malignant condition, having undergone surgery since admission, having an indwelling invasive device on the day of the survey, and particularly a catheter. The four main sites of healthcare-associated infections in acute care hospitals, accounting for 71.5% of documented infection sites—urinary tract (28.5%), surgical sites (15.9%), pneumonia (15.6%), and bacteremia (11.4%)—were identical in 2012 and 2017. However, the proportion of surgical site infections (SSIs) among all infections increased in 2017 compared to 2012, surpassing that of pneumonia. The four most common microorganisms were Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa, accounting for half of the microorganisms isolated from HAI. The decrease in the prevalence of patients infected with methicillin-resistant Staphylococcus aureus (MRSA) between 2012 and 2017 confirms the decline in MRSA infections already observed by the ENP since 2001. The prevalence of patients infected with Enterobacteriaceae resistant to third-generation cephalosporins (3GC) remained stable between 2012 and 2017; that of patients infected with extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-producing Enterobacteriaceae) increased over the period. These results underscore the need to continue prevention efforts targeting the most common and severe infections, particularly surgical site infections (SSIs), bacteremias, and pneumonia.