Abstract Background Healthcare-acquired infections (HAI) in neonatal intensive care unit (NICU) specifically refer to infections that occur in newborn infants within the healthcare environment. Preterm infants, often more vulnerable to infections, in the NICU may be exposed to various medical interventions, devices, and environments that can increase the risk of infections. This systematic review aims to evaluate the effectiveness of interventions proposed to prevent neonatal healthcare-associated infections. Methods PubMed, Scopus and Web of Science databases were searched to identify any RCT study coming from high income countries, published from 2004, that investigated the efficacy of any intervention to prevent HAI in NICU. All type of HAI were considered. Results A total of seven studies were included (2 from USA, 5 from UE). Three studies focused on: administration of donor milk; probiotic Bifidobacterium breve; intravenous Staphylococcus aureus immune globulin; all failed to demonstrate any protective action. Of the four remaining studies that investigate different aspect of the vascular catheterization process only two lead a a statistical significant reduction of HAI. Garland et al. demonstrate in 85 neonates how vancomycin-heparin lock solution is capable to reduce catheter related blood stream infection (CRBSI) 8/43 neonates (18.6%) in the control group vs 0/42 in the vancomycin-heparin-lock group (P = 0.006). Bertini et al. demonstrate in 86 preterm infants how utilization of AgION catheter in Umbelical vein catheterization (UVC) lead to CRBSI rate reduction (2 vs. 22%; p = 0.005), shorter hospital stay (p = 0.04), lower case fatality rate due to Blood stream infection (p = 0.01). Conclusions Among the proposed methodology only the one acting on the catheterization process provide a statistical significant reduction of HAI onset. Widespread observation of proposed protocol may results in reduction of HAI in NICU. Key messages • Modfiication on the catheterization process provide a statistical significant reduction of HAI onset. • Knowing these measures assure reduction of HAI.