<h3>Background</h3> Outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) often occur in neonatal intensive care units (NICUs). However, few studies investigating relevant risk factors and potential interventions have been conducted. The purpose of the current study was to identify risk factors for MRSA infection in NICU patients. <h3>Methods</h3> The current investigation was a prospective cohort study of newborns admitted to the NICU at a Japanese teaching hospital between 1 July 2002 and 31 December 2008. The following patient-related factors were investigated: birth-weight, gestational age, sex, Apgar score, number of fetuses, length until cord detachment, and eye mucous. The medical factors investigated included type of delivery, inborn/outborn, antimicrobial therapy, use of medical devices, Kangaroo mother care, patient-to-nurse ratio, and MRSA colonization pressure. Cox proportional hazards regression was used to analyze risks of MRSA infection, and hazards ratios (HRs) and confidence intervals (CIs) were calculated. The statistical significance level was set at α = 0.05. <h3>Results</h3> A total of 1608 cases were analyzed, and MRSA was isolated from 282 (17.5%) patients. Significant parameters identified in Cox proportional hazards analysis included caesarean section (HR 1.46, 95% CI 1.10–1.95), eye mucous (HR 1.53, 95% CI 1.15–2.05), administration of the mother's first milk via catheter (HR 1.57, 95% CI 1.03–2.41), umbilical catheter placement (HR 2.05, 95% CI 1.02–4.10), patient-to-nurse ratio (HR 0.12, 95% CI 0.05–0.26), and MRSA colonization pressure (HR 16.79, 95% CI 8.41–33.50). <h3>Conclusions</h3> This study identified the risk factors for MSRA infection and determined that identifying high-risk patients, shortening periods of catheterization, maximizing patient-to-nurse ratios, and reducing MRSA colonization pressure can help reduce MRSA transmission among NICU patients.