Objective: Nosocomial infections (NI) are a frequent and important cause of morbidity and mortality in newborn infants who receive intensive care. We sought to determine if comprehensive infection control (CIC) measures decrease rates in a large neonatal intensive care nursery. Methods: Single center interventional study. The CIC intervention consisted of increasing nursing and physician education and awareness of infection rates, establishing common improvement goals, training in hand and environment care, and implementing a specialty nursing team for central venous and arterial catheter care. Demographic and microbiology information for all infants admitted to the NICU from January 1, 1999 to December 31, 2000 established baseline data. The intervention period was during January and February 2001. The postintervention period was March 1, 2001 to February 29, 2004. The main outcome measure was the rate of blood, cerebrospinal and/or urinary tract bacterial infections per 1000 hospital days. Results: Baseline infection rate was 8.5 per 1000 hospital days. The NI rate fell 26% (P=0.002) from baseline in the first year and 29% (P<0.001) in the second and third years after the CIC intervention. The reduction in total NI was due mostly to a 46% fall in coagulase-negative Staphylococcus infection rate (P<0.001); however, rates of all other organisms also fell by 21% (P=0.05). Conclusions: CIC measures can reduce bacterial and fungal NI rates. This effect has been sustained for 3 years following the intervention. © 2006 Nature Publishing Group. All rights reserved.