Objective: To determine the impact of screening and decolonization on methicillin-resistant (MRSA) infection in a neonatal intensive care unit. Study Design: This is a single-center retrospective cohort study comparing patient characteristics among MRSA-colonized and MRSA-infected infants, rates of MRSA infection before and after screening with targeted decolonization, and MRSA infection among those receiving single or combined decolonization agents. Setting: Texas Children's Hospital Pavilion for Women is a 42-bed level three neonatal intensive care units (NICU) in Houston, TX. Patients: Neonates admitted to the NICU from 2012 to 2022 were included in analysis of MRSA colonization and infection. The gestational age ranged from 22 weeks to 42 weeks. Interventions: The MRSA screening methodology consisted of weekly surveillance PCR or culture on admission until discharge. If positive, infants underwent decolonization consisting of topical intranasal mupirocin, and if meeting the gestational and chronological age-based criteria, topical 2% chlorhexidine wipes and topical intranasal mupirocin. Results: The MRSA colonization rate from 2016 to 2022 was 2.2%. Following the screening and decolonization protocol initiated in 2016, there was a sustained downtrend in the rate of MRSA infection. No MRSA-colonized neonates who received both topical mupirocin and Chlorhexidine gluconate (CHG) developed MRSA infection. Conclusions: We observed a decreased rate of MRSA infection in the NICU following implementation of an MRSA screening and decolonization protocol. While our data suggests that the combination of mupirocin and CHG might prevent infection, further studies are needed due to the low prevalence of MRSA infection in our cohort.