The aim of this study was to examine the impact of a multi-factorial intervention on nosocomial infection in very-low-birthweight infants. Consecutive infants with a birth weight less than 1500 g, born between February 2002 and February 2003, were included in this prospective study. The first six-month period (control) included surveillance of current practice. The intervention began in the seventh month and included: (i) changes to handwashing solutions with hand hygiene education; (ii) standardization of intravascular device (IV) insertion with specialized packs; (iii) changes to skin antiseptic solutions (2% aqueous chlorhexidine and 1% chlorhexidine in ethanol); and (iv) mandatory removal or replacement of peripheral IV after 48 hours and removal once enteral intake was > 120 mL/kg/day. Demographic data and details of every device were collected prospectively. Bloodstream infections (BSIs), length of stay (LOS), length of ventilation (LOV) and death were recorded and the rate of nosocomial BSI was calculated. Overall, 174 newborns required 1359 devices. The two cohorts were similar for birth weight and gestation. There was a reduction in nosocomial BSIs from 21% to 9% (control vs. intervention) (P = 0.05, confidence intervals 0.19-1.0). There was no significant difference in LOS, LOV, or mortality. Four infants had complications from 2% chlorhexidine. In conclusion, implementation of the multi-factorial prevention strategy reduced nosocomial BSIs. Alternative antiseptic solutions are needed to reduce the complications caused by 2% aqueous chlorhexidine.