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Introduction Burkholderia pseudomallei is a Gram-negative saprophytic bacillus that causes melioidosis. The infection is endemic in southeast Asia and northern Australia. B. pseudomallei has been designated as a bioterrorism agent and its manipulation should be carried out in a biological safety level 3 laboratory. Workers in laboratories may be accidentally exposed to B. pseudomallei before it is identified, with a risk of laboratory-acquired melioidosis. Objectives We want to describe a case of melioidosis that occurred in our hospital and its management in the laboratory. The objective of this article is to provide guidance to microbiologists confronted with a suspected case of B. pseudomallei. Conclusions We detail a couple of microbiological arguments that can usually guide microbiologists toward presumptive identification of B. pseudomallei. This case report shows the importance of accurate MALDI-TOF MS databases for ensuring accurate microbial identification and antibiotic prophylaxis adapted to exposed individuals. We also want to highlight the importance of developing an effective prevention strategy against any accidental exposure that can occur in a microbiological laboratory.