A prevalence survey is a time-saving and useful tool for obtaining an overview of healthcare-associated infection (HCAI) either in a single hospital or nationally. Direct comparison of prevalence rates is difficult. We evaluated the impact of case-mix adjustment on hospital-specific prevalences. All five tertiary care, all 15 secondary care and 10 (25% of 40) other acute care hospitals took part in the first national prevalence survey in Finland in 2005. US Centers for Disease Control and Prevention criteria served to define HCAI. The information collected included demographic characteristics, severity of the underlying disease, use of catheters and a respirator, and previous surgery. Patients with HCAI related to another hospital were excluded. Case-mix-adjusted HCAI prevalences were calculated by using a multivariate logistic regression model for HCAI risk and an indirect standardisation method. Altogether, 587 (7.2%) of 8118 adult patients had at least one infection; hospital-specific prevalences ranged between 1.9% and 12.6%. Risk factors for HCAI that were previously known or identified by univariate analysis (age, male gender, intensive care, high Charlson comorbidity and McCabe indices, respirator, central venous or urinary catheters, and surgery during stay) were included in the multivariate analysis for standardisation. Case-mix-adjusted prevalences varied between 2.6% and 17.0%, and ranked the hospitals differently from the observed rates. In 11 (38%) hospitals, the observed prevalence rank was lower than predicted by the case-mix-adjusted figure. Case-mix should be taken into consideration in the interhospital comparison of prevalence rates.