Neonates hospitalized in NICU are at risk for healthcare associated infections because of their poor immune defenses, related to gestational age, colonization of mucous membranes and skin with nosocomial microorganisms, exposure to antibiotics, invasive procedures and frequent contacts with healthcare workers (HCWs). Healthcare associated infections are the major source of morbidity and mortality in NICU in the developed world. Most infections are caused by Gram-positive organisms, fulminant sepsis are often associated to Gram-negative organisms, fungal sepsis occurs frequently in ELBW infants. Hand hygiene is the most important preventive procedure, nevertheless hand hygiene compliance among HCWs remains low. Continuous educational strategies can improve hand hygiene and contribute to reducing the incidence of neonatal infections. Other important prevention strategies include early enteral feeding with human milk, minimization and safety in the use of invasive devices, limiting unnecessary empiric broadspectrum antibiotics, eventual use of lactoferrin bifidobacteria and lactobacilli, prophylactic administration of fluconazole in VLBW. Emergence of multi drug resistant organisms (MDRO) is a worrying perspective. Methicillin-resistant Staphylococcus aureus (MRSA) is an important healthcare-associated pathogen. Active surveillance culturing for MRSA carriers, in combination with contact precautions and decolonization in some hyperendemic settings, has been proved to reduce MRSA transmission and infection rates. Multidrug-resistant Gram-negatives are frequently reported. Overuse of antimicrobial drugs and crosstransmission via caregiver hands, contaminated equipment or inanimate objects are the major drivers of selection and dissemination. Strategies to control outbreaks of MDRO colonization/infection in the NICU may include performing hand hygiene, cohorting and isolating patients, screening healthcare workers and performing admission and periodic surveillance cultures.