Objective To investigate a suspected outbreak of hospital-acquired infections caused by Mycobacterium chelonae related to flexible bronchoscope (hereinafter referred to as “bronchofibroscope”) and apply targeted high-throughput sequencing (tNGS) technology for etiological analysis, providing references for controlling hospital infection outbreaks. Methods A retrospective survey of patients who were detected with Mycobacterium chelonae through tNGS testing of bronchoalveolar lavage fluid (BALF) after bronchofibroscopy at the Zhengdong District, People’s Hospital of Henan University of Chinese Medicine, People’s Hospital of Zhengzhou between May 1, 2018 and March 18, 2024. The causes were investigated through comprehensive measures including on-site epidemiological surveys and environmental health assessments, and intervention measures were developed and evaluated for effectiveness. Results A total of 52 patients were included. Mycobacterium chelonae was detected in 30 patients, nosocomial infection was excluded in all cases. The suspicious contaminated bronchofibroscope lavage fluid and its cleaning and disinfection equipment, environment and other samples were collected. The traditional microbial culture results were negative. The tNGS results showed that Mycobacterium chelonae was detected in bronchofibroscope lavage fluid (sequence number 156), and all the patients with Mycobacterium chelonae detected in BALF used the bronchofibroscope. It was judged that this event was a pseudo-outbreak of nosocomial infection caused by the contamination of bronchofibroscope with the patient’s BALF. After three months of continuous follow-up after the comprehensive control measures were taken, Mycobacterium chelonae was not detected by tNGS in bronchofibroscope lavage fluid or patients’ BALF. All patients in the hospital improved and discharged without any new cases. The pseudo-outbreak of nosocomial infection was effectively controlled. Conclusions There are many links in the reprocessing of bronchofibroscope, which is easy to cause pollution, and the management needs to be strengthened. tNGS detection has the characteristics of high efficiency, few background bacteria and clear pathogen spectrum, which can be used as a supplementary means for the investigation of nosocomial infection outbreaks, and is of great significance for identifying the source of infection and determining the transmission route.