This study analysed daily antimicrobial costs of Intensive Care Unit (ICU)-acquired, laboratory-confirmed bloodstream infection (BSI) per patient admitted to the ICU of a university hospital, based on prospectively collected data over a 4-year period (2003-2006). Costs were calculated based on the price of the agent(s) initiated on the first day of appropriate treatment and according to: (i) focus of infection; (ii) pathogen; and (iii) antimicrobial agent. The study included 310 adult patients who developed 446 BSI episodes. Mean overall daily antimicrobial cost was euro114.25. Daily antimicrobial cost was most expensive for BSIs with unknown focus (euro137.70), followed by catheter-related (euro122.73), pulmonary (euro112.80), abdominal (euro98.00), wound (euro89.21), urinary (euro87.85) and other inciting focuses (euro81.59). Coagulase-negative staphylococci were the most prevalent pathogens isolated. Treatment of BSIs caused by Candida spp. was the most costly. The daily antimicrobial costs per infected patient with multidrug-resistant BSI was ca. 50% higher compared with those without (euro165.09 vs. euro82.67; P<0.001). Among the total of 852 prescriptions, beta-lactam antibiotics accounted for approximately one-third of the overall daily cost of antimicrobial agents. The antibiotic cost associated with ICU-acquired, laboratory-confirmed BSI is significant and should be reduced by implementing infection control measures and preventive strategies.