Objective To analyze the causes and intervention measure efficacy of an outbreak of neonatal Pseudomonas aeruginosa (P. aeruginosa) infection in a medical institution. Methods The epidemiological investigation was conducted on 5 neonates who had P. aeruginosa infection in the neonatal intensive care unit (NICU) of this medical institution from May 26 to June 10, 2024. Specimens were taken from potentially contaminated environments to infer the cause of P. aeruginosa transmission in the NICU, infection control measures were also taken, and the efficacy of the control measures was continuously monitored. Results Among the 5 infected neonates, 3 had lower respiratory tract infection and 2 had lower respiratory tract combined with bloodstream infection. Seven strains of P. aeruginosa were detected with a consistent antimicrobial resistance spectrum. Infected neonates were in the same ward and had spatial and temporal clustering. Through environmental hygiene monitoring, P. aeruginosa with consistent antimicrobial resistance spectrum of neonatal infection was isolated from the NICU handwashing sink and water-related environment (suction connection pipe, bedside isolation gowns). Infection was controlled after improving sink disinfection methods and enhancing contact isolation measures, and no further P. aeruginosa was detected in the environment. Conclusion The outbreak of neonatal HAI in the NICU is inferred to be caused by contamination of ward handwashing sink by P. aeruginosa, then further contaminated the suction connection tube, thereby leading to the transmission of neonatal infection through suction procedures. Healthcare-associated infection surveillance and control professionals need to fully understand the characteristics of outbreak pathogens and take targeted measures to curb the transmission of neonatal infection.