A one-day survey was conducted in all (19) Slovenian acute-care hospitals in October 2001 to estimate the prevalence of all types of hospital-acquired infections (HAIs) and to identify predominant micro-organisms and risk factors. Among 6695 patients surveyed, the prevalence of patients with at least one HAI was 4.6%. The prevalence of urinary tract infections was highest (1.2%), followed by pneumonia (1.0%), surgical wound infection (0.7%), and bloodstream infection (0.3%). In intensive care units (ICUs) the prevalence of patients with at least one HAI was 26.9% and the ratio of episodes of HAI per number of patients was 33.3%. One or more pathogens were identified in 55.8% of HAIs episodes. Among these, the most frequently single isolated micro-organisms were Staphylococcus aureus (18.2%) and Escherichia coli (10.2%). Risk factors for HAI included central intravascular catheter (adjusted odds ratio (OR) 3.2; 95% confidence intervals (CI) 2.1-4.9), peripheral intravascular catheter (adjusted OR 1.7; 95% CI 1.2-2.4), urinary catheter (adjuster OR 2.4; 95% CI 1.6-3.4), and hospitalization in ICUs (adjusted OR 2.5; 95% CI 1.4-4.3). The results provide the first national estimates for Slovenia.