In this study, we aimed to determine the utility of a multiple system intervention to reduce catheter-related bloodstream infections (CR-BSI) in our intensive care unit (ICU). A prospective cohort study was undertaken in the medical and surgical ICU at a university hospital. We applied five measures: educational sessions about inserting and maintaining central venous catheters, skin cleaning with chlorhexidine, a checklist during catheter insertion, subclavian vein insertion and avoiding femoral insertion whenever possible, and removing unnecessary catheters. We determined the rate of CR-BSI per 1,000 catheter-days during the intervention (March to December 2007) and compared it with the rate during the same period in 2006 in which we applied only conventional preventive measures. CR-BSI was defined as the recovery of the same organism (same species, same antibiotic susceptibility profile) from catheter tip and blood cultures. We registered 4,289 patient-days and 3,572 catheter-days in the control period and 4,174 patient-days and 3,296 catheter-days in the intervention period. No significant differences in the number of patients with central venous catheters during the two periods were observed: catheters were used in 81.5% of patients during the control period and in 80.6% of patients during the intervention period. During the control period, 24 CR-BSI were diagnosed (6.7/1,000 catheter-days); during the intervention period, 8 CR-BSI were diagnosed (2.4/1,000 catheter-days) (relative risk 0.36; 95% confidence interval [CI] 0.16 to 0.80; p = 0.015). Nurses interrupted the procedure to correct at least one aspect when completing the checklist in 17.7% of insertions. In conclusion, a multiple system intervention applying evidence-based measures reduced the incidence of CR-BSI in our ICU.