The aim of this study was to determine, over time, changes in annual trends of nosocomial bacteraemia (NB) and to quantify pathogen-specific changes and emergence of antibiotic resistance. A retrospective cohort study in a 997 bed tertiary care centre in the Netherlands was performed. All adult patients (>= 18 years old) admitted for > 48 h between 1 January 1996 and 31 December 2005 were included. A total of 163 525 patients, comprising 1 826 852 patient-days and 1785 episodes of NB, were analysed. The number of admissions per year and length of hospital stay decreased over time. Crude incidence of NB per year remained unchanged, but cumulative incidence (cases/10 000 admissions) and incidence densities (cases/100 000 patient-days at risk) increased, on average, by 2.0% and 4.0% per year, respectively, primarily because of infections caused by Enterococcus spp. and Pseudomonas aeruginosa. The incidence density of NB caused by highly resistant microorganisms increased, on average, by 26.1% [95% confidence interval (CI): 17-37] per year, when compared with an annual increase of 3% (95% CI: 1-5) for NB caused by susceptible pathogens. Ratios of increased incidence densities of resistant and susceptible bacteria were 8.7, 3.5, 2.6 and > 37.9 for all pathogens, Enterococcus spp., P. aeruginosa and Enterobacteriaceae, respectively. Due to changes in the patient population, increased incidences of NB over time are only evident when expressed as cumulative incidence or incidence densities. Despite overall low levels of antibiotic resistance, the incidence of NB caused by multiresistant pathogens rapidly increased, adding to the total burden of NB.