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Background: Vancomycin-resistant Enterococcus faecium (VRE) may cause outbreaks in neonatal intensive care units (NICU). We describe a biphasic VRE outbreak and identify risk factors for VRE acquisition., Methods: After the occurrence of 2 cases of VRE infections in a 44-bed NICU, a bundle of interventions was implemented that included active surveillance cultures for VRE, enhanced infection control measures, and audits on antimicrobial use, from June to December 2008. Analysis was performed using polymerase chain reaction and pulse-field gel electrophoresis techniques. A case-control study was conducted to identify risk factors., Results: Among 253 neonates screened, 101 (39.9%) were found to be colonized with VRE. During the first 9 weeks of the study period, 59 new cases were detected. Molecular analysis showed 1 predominant clone. During weeks 10-12, no new cases of VRE colonization were detected; however, at week 13, just when the outbreak appeared to be over, a second wave occurred, with 42 new cases and multiple clones detected. Multivariate analysis identified administration of antimicrobial therapy for late-onset neonatal sepsis and hospitalization during the first month of this outbreak as significant risk factors for VRE colonization., Conclusion: Both a high prevalence of VRE colonization and antimicrobial use promoted the transmission of VRE during this biphasic outbreak. Adherence to infection control measures and antimicrobial stewardship policies are of utmost importance., (Copyright © 2013 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.)