PURPOSE: We aimed to assess the incidence of methicillin-resistant Staphylococcus aureus (MRSA) colonization and infection in a neonatal intensive care unit (NICU) by using various decolonization methods and to evaluate their efficacy. METHODS: Medical records of all neonates who were admitted to the NICU of Seoul National University Children's Hospital were retrospectively reviewed. Surveillance culture were obtained for all neonates in the NICU 48 hours after admission. Three periods with different decolonization methods were compared; Period 1 was without any decolonization measures (July 1, 2009 to August 26, 2010). In period 2, intranasal mupirocin and chlorhexidine gluconate bathing were administered to MRSA-colonized neonates (August 27, 2010 to September 6, 2011). In period 3, only chlorhexidine bathing was performed for MRSA-colonized infants (September 7, 2011 to August 31, 2012). RESULTS: A total of 1,378 infants were admitted to the NICU during the study period. Baseline demographic and clinical characteristics were similar among the 3 periods. The incidence of MRSA colonization per 1,000 patient-days was 6.27 for period 1, 7.02 for period 2, and 6.29 for period 3; however, these values were not significantly different. The incidence of MRSA infection was highest in period 3, with 0.69 cases per 1,000 patient-days; however, this finding was not significant. The MRSA infection/colonization ratio also did not differ significantly among the 3 study periods. CONCLUSION: Decolonization of MRSA in the NICU with the application of chlorhexidine gluconate bathing alone or in combination with intranasal mupirocin were not effective in decreasing the incidence of MRSA colonization and infection.