Background <p>In July 2023, <i>Burkholderia gladioli</i> was detected in a patient’s ascites specimen. This pathogen had not been detected in the hospital for nearly 3 years. Therefore, an investigation was immediately launched.</p> Methods <p>Microbial environmental hygiene samples were collected from the operating room environment, the ultrasonic probe, and ultrasonic coupling agents in the interventional ultrasound department. The quantitative method was used to culture and identify bacterial species. Average Nucleotide Identity was used to compare the similarity of all orthologous protein-coding genes between pairwise genomes.</p> Results <p>The antimicrobial susceptibility spectrum of <i>B. gladioli</i> isolated from the coupling agent was consistent with that of strains isolated from the patient sample. The two isolates were the same clone and 100% identical according to ANI. The qualified rate of non-sterile coupling agent was 18.37%; sterile coupling agents were 100% qualified. With contaminated and qualified ultrasonic coupling agent, the number of bacteria exceeding the standard rate was 88.00% and 56.00%, respectively. The qualified rate of total bacterial count of the ultrasonic probe disinfected with disinfectant wipes or ultraviolet light reached 90% or 100%, respectively. The rate of carbapenem-resistant <i>Acinetobacter baumannii</i> and carbapenem-resistant <i>Klebsiella pneumoniae</i> detected on the bedside ultrasound machine was 13.04% (6/46).</p> Conclusion <p>In this case, we identified contaminated ultrasonic coupling agent and irregular use of non-sterile coupling agent. To avoid coupling agent contamination, monitoring and routine cleaning of the ultrasonic examination environment are crucial. Robust environmental testing and epidemiological investigation, along with early identification of uncommon bacteria using information system monitoring, are essential to contain an outbreak.</p> Clinical trial number <p>Not applicable.</p>