•Burkholderia gladioli was identified as a cause of healthcare-associated bacteremia.•The investigation traced to the contaminated multidose vials with saline solution.•Isolates from blood and saline showed the same susceptibility patterns.•Pulsed-field gel electrophoresis (PFGE) was used to genotype the strains.•PFGE profile of all B. gladioli isolates demonstrated clonal relatedness. Burkholderia gladioli has been associated with infections in patients with cystic fibrosis, chronic granulomatous disease, and other immunocompromising conditions. The aim of this study was to better depict the outbreak of healthcare-associated bacteremia caused by B. gladioli due to exposure to contaminated multidose vials with saline solutions. An environmental and epidemiologic investigation was conducted by the Infection Prevention and Control Team (IPCT) to identify the source of the outbreak in three Croatian hospitals. During a 3-month period, 13 B. gladioli bacteremia episodes were identified in 10 patients in three Croatian hospitals. At the time of the outbreak, all three hospitals used saline products from the same manufacturer. Two 100-ml multidose vials with saline solutions and needleless dispensing pins were positive for B. gladioli. All 13 bacteremia isolates and two isolates from the saline showed the same antimicrobial susceptibility patterns and pulsed-field gel electrophoresis profile, demonstrating clonal relatedness. When an environmental pathogen causes an outbreak, contamination of intravenous products must be considered. Close communication between the local IPCT and the National Hospital Infection Control Advisory Committee is essential to conduct a prompt and thorough investigation and find the source of the outbreak. Display omitted