Objective To investigate the causes of a clustering event of neonatal septicemia in the neonatal intensive care unit(NICU) of a hospital, evaluate the effectiveness of relevant control measures. Methods The preterm triplets with healthcare-associated neonatal septicemia in NICU of a hospital from April 23 to 25, 2019 were performed epidemiological survey, specimens of breast milk and hospital environment were collected for hygienic monitoring, and control measures were taken for the existing risk factors. Results There was no other neonatal septicemia case except triplets in NICU. Triplets all developed disease 48 hours after the start of breastfeeding, 2 neonates were isolated Enterobacter cloacae from blood culture. Among the 37 collected specimens, bacterial colony number of 4 specimens exceeded the standard, Enterobacter cloacae was only isolated from breast milk, and the colony number was>1 000 CFU/mL. Breastfeeding of triplets was suspended temporarily, antimicrobial treatment was given, and management of various infection prevention and control measures was strengthened, triplets were all cured and discharged from hospital, there was no new case. Conclusion Breast milk contamination may be the cause of this clustering event. In order to minimize the risk of healthcare-associated infection, standardized management of breastfeeding should be implemented in neonatal department.