Background: The prophylactic use of antimicrobial agents to prevent infections in non-surgical situations has hardly been investigated. We investigate the extent, indications and appropriateness of antimicrobial prophylaxis given outside the operating room in a tertiary care hospital. Methods: Four point-prevalence surveys were conducted in which all inpatients on that day were screened for the use of prophylactic antimicrobials: medical prophylaxis, prophylaxis around non-surgical interventions and surgical prophylaxis given on the ward. The primary endpoint was the extent of prophylaxis relative to the total number of antimicrobial prescriptions. We also investigated per prescription the presence of a (local) protocol and adherence to these protocols. Results: We registered in total 1020 antimicrobial prescriptions, of which 317 (31.1%) were given as prophylaxis. 827/ 1020 were antibiotic prescriptions. Of these antibiotic prescriptions, 17.0% was medical prophylaxis, 2.7% prophylaxis around non-surgical interventions and 6.9% surgical prophylaxis administered on a ward. For medical antibiotic prophylaxis, a protocol was present in 125 of 141 prescriptions (88.7%); the protocol was followed in 118 cases (94.4%). For prophylaxis around non-surgical interventions and surgical prophylaxis on the wards, protocol presence and adherence rates were 59.1% and 92.3%, and 73.3% and 97.6% respectively. Of the 96 antiviral and 97 antifungal prescriptions, 42.7% and 57.8%, respectively, were medical prophylaxis, of which 95.1 and 96.3% were prescribed according to protocols respectively. Conclusions: Antimicrobial prophylaxis outside the operating theatre is responsible for a considerable part of total in-hospital antimicrobial use. For most prescriptions there was a protocol and adherence to the protocols was high. The main targets for improvement were prophylaxis around non-surgical interventions and surgical prophylaxis given on the ward.