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Infants hospitalized in the neonatal intensive care unit, particularly preterm infants, have very high rates of nosocomial sepsis (also referred to as late onset sepsis or healthcare-associated sepsis). Today's preventive strategies for nosocomial sepsis focus on augmenting the immunologic and functional immaturities of premature infants and ameliorating the risks of extrinsic factors by the use of prophylactic antibiotics and best clinical practices.Topical emollients improved neonatal skin condition, but were associated with an increased risk of nosocomial bacterial sepsis and coagulase negative staphylococcal infections, and thus should not be used in extremely-low-birth-weight infants. Single-center studies have shown that probiotics containing anaerobic bacteria may reduce the rate of necrotizing enterocolitis, the severity of necrotizing enterocolitis, and/or bacterial sepsis. Single-center studies have shown that prophylactic fluconazole reduces the rates of invasive candidiasis and/or colonization of extremely-low-birth-weight infants. Quality improvement projects to improve adherence to appropriate hand hygiene and best practices for central venous catheter insertion and maintenance can reduce rates of nosocomial sepsis.The safety and efficacy of probiotics and prophylactic fluconazole require large multicenter trials. Quality improvement initiatives, however, can be performed now and can reduce the rates of nosocomial sepsis in the neonatal intensive care unit.