Objective To evaluate the status and risk factors of healthcare-associated infection (HAI) after ente-rostomy in neonates. Methods Basic information, clinical and postoperative HAI data of neonates underwent ente-rostomy from June 2019 to June 2022 in the department of neonatal surgery of a hospital were collected. Neonates were divided into the HAI group and non-HAI group according to whether HAI occurred after operation. The HAI-related risk factors were analyzed by univariate analysis and logistic regression. Results A total of 227 neonates underwent surgery were analyzed, with 71 neonates having 84 episodes of HAI and a HAI incidence of 31.28%. Infection sites were mainly surgical site infection (n=24), gastrointestinal infection (n=24) and bloodstream infection (n=23). 49 pathogenic bacterial strains were detected in HAI cases, mainly Gram-positive bacteria (59.18%). Univariate analysis showed statistically significant differences between two groups in gestational status, gestational age at birth, birth weight, primary disease, fistula location, fistula mode, postoperative ventilator use, and postoperative fasting time(all P < 0.05). Binary logistic multivariate regression analysis showed that low gestational age at birth (OR=0.806, 95%CI[0.676-0.962]) and neonatal necrotizing enterocolitis (NEC) (OR=0.484, 95%CI[0.247-0.948]) were risk factors for the development of HAI after enterostomy in neonates (both P < 0.05). Conclusion Low gestational age at birth and NEC are risk factors for the postoperative HAI in neonates underwent enterostomy. Clinical attention should be paid to these neonates.