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Objective: To investigate the inhibitory effect on Burkholderia pseudomallei (B. pseudomallei) strain HNBP001 of a bacillomycin D-like cyclic lipopeptide compound named bacillomycin DC isolated from Bacillus amyloliquefaciens HAB-2. Methods: The antibacterial effect of bacillomycin DC on B. pseudomallei was determined using the disk diffusion method. The minimum inhibitory concentrations were evaluated by microdilution assay. In addition, transmission electron microscopy was performed and quantitative real-time polymerase chain reaction assay was carried out to determine the expression of MexB, OprD2, and qnrS genes. Results: Bacillomycin DC produced an inhibition zone against B. pseudomallei with minimum inhibitory concentration values of 12.5 μg/mL 24 h after treatment and 50 μg/mL at 48 and 72 h. Transmission electron microscopy showed that bacillomycin DC resulted in roughening cell surface and cell membrane damage. Quantitative real-time polymerase chain reaction analysis showed low expression of MexB, OprD2 and qnrS genes. Conclusions: Bacillomycin DC inhibits the growth of B. pseudomallei and can be a new candidate for antimicrobial agents of B. pseudomallei.