A survey was carried out to determine the prevalence and appropriateness of antimicrobial therapy (AMT) in the Netherlands and to identify determinants for inappropriate AMT. Prevalence surveys of patients hospitalised in the Netherlands were performed three times in 2008 and 2009. Patients' demographic, infection-related and AMT-related data were collected from hospital wards. A total of 19 hospitals participated, consisting of a mix of university, teaching and general hospitals, which were distributed evenly across the country. The appropriateness of AMT was assessed using a standardised algorithm based on local AMT prescription guidelines. A total of 7,853 patients were included, of which 2,327 (29.6%) patients were on AMT (range: 20.8–39.5%). In 372 patients (16% of patients on AMT), treatment was considered inappropriate. In 265 (11.4%) patients on AMT, appropriateness of treatment was not judged because of insufficient information. The percentage of patients without a judgment varied considerably between the participating hospitals (range: 1.3–36.2%). Appropriate AMT use was significantly associated with a patient being in an intensive care unit, having a central venous catheter and being given beta-lactamase-sensitive penicillins. The use of fluoroquinolones was significantly associated with more frequent inappropriate use. There was considerable and significant variation between the participating hospitals in the amount of antimicrobials prescribed and the appropriateness of their use. To improve the completeness and reliability of such surveys, there is a need for intensive training of observers and medical staff in recording information.