Objective To understand the spectrum and changes of pathogens causing healthcare-associated infection (HAI) in neonatal intensive care unit (NICU). Methods Clinical medical records of neonates with HAI in a hospital from January 2018 to December 2022 were collected, spectrum of pathogens causing HAI were and analyzed retrospectively. Results A total of 7 597 hospitalized neonates were investigated, and 240 of whom had 263 cases of HAI, with an HAI incidence of 3.16% and healthcare-associated case infection incidence of 3.46%. 96 cases (36.50%) were bloodstream infection, 70 (26.62%) were respiratory system infection, and 57 (21.67%) were infection without clear sites. A total of 170 pathogens were detected from specimens, 78 (45.88%) of which were Gram-positive bacteria, with Staphylococcus spp. accounting for the highest proportion, 78 (45.88%) were Gram-negative bacteria, mainly Klebsiella pneumoniae, and 14 (8.24%) were fungi. The detection rate of Gram-negative bacteria showed an upward trend from 2018 to 2022 (P<0.01). Conclusion The majority of HAI in NICU is bloodstream infection. In recent years, the detection rate of Gram-negative bacteria has been increasing year by year, and it is necessary to streng-then the prevention and control of HAI in clinical practice.