Objective To explore the application of metagenomic next-generation sequencing (mNGS) technology in the investigation of healthcare-associated infection (HAI) outbreaks of carbapenem-resistant Acinetobacter baumannii (CRAB). Methods Pathogenic detection by mNGS and conventional pathogen culture were performed on 5 patients in the intensive care unit (ICU) of a hospital from June 8 to 22, 2023 from whom CRAB were detected. Microbial sampling was carried out in potentially contaminated environment. Bacterial culture, identification, and antimicrobial susceptibility testing were conducted. Comprehensive control measures were taken, and the effect was evaluated. Results The time required for reporting results by mNGS was shorter than the culture time ([3.92 ± 1.05] days vs[6.24 ± 0.25] days, P<0.001). CRAB was isolated from the specimens of 5 patients. mNGS detected OXA-23 resistance genes from all patients. After comprehensive assessment by experts, 4 patients were HAI and 1 patient was due to specimen contamination. According to the definition from Guidelines for HAI outbreak control, this event was considered an outbreak of HAI. The monitoring results of environmental hygiene showed that the detection rate of CRAB in the environment during the outbreak was 51.30% (59/115), mainly from the hands of health care workers and the surface of ventilators. After implementing multidisciplinary infection control measures, clinicians' hand hygiene compliance rate and implementation rate of ventilator disinfection increased from 40.83% (49/120) and 33.33% (16/48) to 82.61% (95/115) and 83.33% (30/36), respectively. The prognosis of patients was good, and no new case emerged during subsequent monitoring. The outbreak of HAI in this hospital has been effectively controlled. Conclusion mNGS is characterized by high precision, less time consumption, and high accuracy, and can be applied to the prevention and control of HAI outbreak and the study of antimicrobial-resistant genomes. It is of great significance for the anti-infection treatment of patients with multidrug-resistant orga-nism infection as well as the formulation of HAI prevention and control measures. Continuous improving disinfection effectiveness and hand hygiene compliance is important for preventing and controlling CRAB infection.