Objectives To report and analyse trends in antibiotic use in Dutch university hospitals, large teaching hospitals and general hospitals over the period 2003 to 2009. Methods Data on the use of antibiotics and hospital resource indicators were obtained by distributing a questionnaire to all Dutch hospital pharmacies. Antibiotic use was expressed as the number of defined daily doses (DDDs) per 100 patient-days, per 100 admissions and per 1000 inhabitants per day. The latter was achieved by extrapolating sample data by means of imputation and up-scaling. Results From 2003 to 2009, the mean length of hospital stay decreased from 6.27 to 4.50 days (−28%). Total systemic antibiotic use significantly increased from 52.3 to 69.8 DDDs per 100 patient-days ( P < 0.001). Despite the overall constant use when expressed in DDDs per 100 admissions, we found a significant increase in the total use of piperacillin/tazobactam, cefazolin, ceftriaxone, meropenem, azithromycin, gentamicin, ciprofloxacin and vancomycin. Mean total systemic use expressed in DDDs per 1000 inhabitants per day gradually increased by 38% from 0.73 in 2003 to 1.01 in 2009. Conclusions Total hospital antibiotic consumption is still low in the Netherlands compared with other European countries. Also, between 2003 and 2009 the use of antibiotics in individual hospitalized patients remained stable. However, since they remained in the hospital for a shorter period of time, the number of DDDs per 100 patient-days increased. This results in an intensification of antibiotic treatment per hospital bed, leading to a possible increase in selection pressure towards resistance. This may create a problem for future patients. To limit the emergence and transmission of antimicrobial-resistant bacteria, effective antibiotic stewardship is essential.