IntroductionThis study aimed to assess the role of adenosine triphosphate (ATP) bioluminescence level monitoring for identifying reservoirs of the outbreak pathogen during two consecutive outbreaks caused byEnterococcus faeciumandStaphylococcus capitisat a neonatal intensive care unit (NICU). The secondary aim was to evaluate the long-term sustainability of the infection control measures employed one year after the final intervention measures.MethodsTwo outbreaks occurred during a 53-day period in two disconnected subunits, A and B, that share the same attending physicians. ATP bioluminescence level monitoring, environmental cultures, and hand cultures from healthcare workers (HCW) in the NICU were performed. Pulsed-field gel electrophoresis (PFGE) typing was carried out to investigate the phylogenetic relatedness of the isolated strains.ResultsFour cases ofE. faeciumsepsis (patients A-8, A-7, A-9, B-8) and three cases ofS. capitissepsis (patients A-16, A-2, B-8) were diagnosed in six preterm infants over a span of 53 days. ATP levels remained high on keyboard 1 of the main station (2076 relative light unit [RLU]/100 cm2) and the keyboard of bed A-9 (4886 RLU/100 cm2). By guidance with the ATP results, environmental cultures showed thatE. faeciumisolated from the patients and from the main station’s keyboard 1 were genotypically indistinguishable. Two differentS. capitisstrains caused sepsis in three patients. A total 77.8% (n = 7/9) ofS. capitiscultured from HCW's hands were genotypically indistinguishable to the strains isolated from A-2 and A-16. The remaining 22.2% (n = 2/9) were genotypically indistinguishable to patient B-8. Three interventions to decrease the risk of bacterial transmission were applied, with the final intervention including a switch of all keyboards and mice in NICU-A and B to disinfectable ones. Post-intervention prospective monitoring up to one year showed a decrease in blood culture positivity (P= 0.0019) and catheter-related blood stream infection rate (P= 0.016) before and after intervention.ConclusionATP monitoring is an effective tool in identifying difficult to disinfect areas in NICUs. Non-medical devices may serve as reservoirs of pathogens causing nosocomial outbreaks, and HCWs' hands contribute to bacterial transmission in NICUs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13756-023-01273-5.