A retrospective cohort study on nosocomial infections (NI) in the neonatal intensive care unit (NICU) was performed in the Children's Hospital of Zhejiang University, Hangzhou district, China. The most common infection site was pneumonia and bloodstream infection. Low admission age, long NICU stay, and mechanical ventilation were significant risk factors for NI. Klebsiella pneumoniae was the most common pathogen, followed by Acinetobacter baumannii, Staphylococcus epidermidi, Pseudomonas aeruginosa, Enterobacter cloacae, and Stenotrophomonas maltophilia. Antibiotic resistance of the isolated bacterium was high. In conclusion, this study described the clinical characteristics of NI in a Chinese NICU, which might contribute to implementation of more effective therapeutic and preventive strategies.