Infants cared for in the neonatal intensive care unit (NICU) are at risk for nosocomial infection. Immature immune systems and dependence on indwelling supportive devices means that sick neonates are at particularly high risk. Prevention of horizontal transmission of—and subsequent infection with—pathogens within the NICU requires a multifaceted approach. Minimizing entry of pathogens into the NICU, close attention to standard and transmission-based precautions, and care bundles for medical devices can reduce nosocomial infections. In this chapter, NICU-associated infections are presented, and prevention strategies are highlighted.