Background. Methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of morbidity and mortality among neonates admitted to neonatal intensive care units (NICUs). The MRSA colonization of neonates, attributed to various sources, including mothers, healthcare workers, and environmental surfaces, can lead to severe infection, prolonged hospital stays, and even death, imposing substantial economic burdens. Given the pressing need to mitigate MRSA spread in these vulnerable environments, further examination of the subject is warranted. This systematic review is aimed at synthesizing available evidence on MRSA carriage proportions among mothers of newborns, healthcare workers, and environmental surfaces in NICUs. Methodology. We included observational studies published in English or French from database inception to March 21, 2023. These studies focused on MRSA in nonoutbreak NICU settings, encompassing healthy neonate mothers and healthcare workers, and environmental surfaces. Literature search involved systematic scanning of databases, including Medline, Embase, Web of Science, Global Health, and Global Index Medicus. The quality of the selected studies was assessed using the Hoy et al. critical appraisal scale. The extracted data were summarized to calculate the pooled proportion of MRSA positives, with a 95% confidence interval (CI) based on the DerSimonian and Laird random-effects model. Results. A total of 1891 articles were retrieved from which 16 studies were selected for inclusion. Most of the studies were from high-income countries. The pooled proportion of MRSA carriage among 821 neonate mothers across four countries was found to be 2.1% (95% CI: 0.3-5.1; I2=76.6%, 95% CI: 36.1-91.5). The proportion of MRSA carriage among 909 HCWs in eight countries was determined to be 9.5% (95% CI: 3.1-18.4; I2=91.7%, 95% CI: 87.1-94.6). The proportion of MRSA carriage among HCWs was highest in the Western Pacific Region, at 50.00% (95% CI: 23.71-76.29). In environmental specimens from five countries, a pooled proportion of 16.6% (95% CI: 3.5-36.0; I2=97.7%, 95% CI: 96.6-98.4) was found to be MRSA-positive. Conclusion. With a significant heterogeneity, our systematic review found high MRSA carriage rates in neonate mothers, healthcare workers, and across various environmental surfaces in NICUs, posing a potential risk of nosocomial infections. Urgent interventions, including regular screening and decolonization of MRSA carriers, reinforcing infection control measures, and enhancing cleaning and disinfection procedures within NICUs, are crucial. This trial is registered with CRD42023407114.