Objective To investigate the bacterial colonization and disinfection effect of infusion connectors in neonatal intensive care unit (NICU), and provide clinical reference for improving the safety management of intravenous infusion in neonates. Methods 59 infusion connectors in NICU were selected as the control group. 52 disinfected infusion connectors during the same period were selected as the observation group A, meanwhile, disinfection time and method used by nurses were observed. After training on disinfection method, 50 disinfected infusion connectors were selected as the observation group B. All infusion connectors were sampled for culture. Results In the control group, bacteria were detected from 53 connectors, with a detection rate of 89.8%. Among them, 39 connectors were isolated 1 bacterial specie respectively, 13 were isolated 2 bacterial species respectively and 1 was isolated 3 bacterial species. Bacteria were detected from 13 connectors in observation group A, and the detection rate was 25.0%. In observation group B, bacteria were detected from 4 connectors, with a detection rate of 8.0%. Diffe-rence in the positive rates of bacterial culture before and after training was statistically significant(χ²=84.418, P < 0.001). Conclusion Infusion connectors in NICU are prone to be contaminated. Effective disinfection can significantly reduce bacterial colonization. Health care workers should be aware of the risks of infusion-associated infection, standardize infusion operation, strengthen the management of infusion devices, and explore more effective disinfection methods.