A point-prevalence survey of five European university hospitals was performed to benchmark antimicrobial drug use in order to identify potential problem areas in prescribing practice and to aid in establishing appropriate and attainable goals. All inpatients at the university hospitals of Rijeka (Croatia), Tartu (Estonia), Riga (Latvia), Vilnius (Lithuania) and Karolinska-Huddinge (Sweden) were surveyed for antimicrobial drug use during a single day. The frequency of antimicrobial drug use was 24% in Rijeka, 30% in Tartu, 26% in Riga, 14% in Vilnius and 32% in Huddinge. Surgical patients were treated with antimicrobial agents more often than medical patients in Riga (53% vs. 31%), Tartu (39% vs. 26%) and Vilnius (54% vs. 25%). Two-thirds of patients in Rijeka, Tartu, Riga and Vilnius, and fewer than half of the patients in Huddinge, received antimicrobial agents intravenously. Broad-spectrum antimicrobial agents were used most commonly in Rijeka. The prevalence of nosocomial infections treated with antibiotics was 9% at Huddinge, and 3–5% at the other centres. Benchmarking antimicrobial drug use at five university hospitals identified differences and problem areas. The high rates of intravenous administration, poor compliance with guidelines, and prolonged surgical prophylaxis were general problems that deserved specific attention at all centres. A change in prescription practices may reduce unnecessary drug use and decrease antimicrobial resistance.