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Staphylococcus aureus (S. aureus) continues to be a leading cause of outbreaks and health-care-associated infections in neonatal intensive care units. In the first few months of life, many neonates acquire S. aureus as part of their delicate and evolving microbiota. Neonates that asymptomatically acquire S. aureus colonization are at increased risk of developing a subsequent S. aureus infection. This review discusses the epidemiology and prevention of S. aureus disease in neonates and how decolonization to eradicate S. aureus may decrease S. aureus transmission and infections in the neonatal intensive care unit.