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Diagnosing Buruli ulcer (BU) is complicated by limited access to the sensitive IS2404 qPCR. Experienced clinicians report a distinct odour of Buruli ulcers. We explored the potential of headspace analysis by thermal desorption-gas chromatography-mass spectrometry to detect volatile organic compounds (VOCs) from Mycobacterium ulcerans both in vitro and clinically. This study was conducted in two phases: a discovery and validation phase. During the discovery phase, VOCs that enable identification of M. ulcerans cultures were determined. During the validation phase, these VOCs were evaluated in clinical samples for which we used gauzes from patients with skin ulcerations in the Democratic Republic of Congo. Seven M. ulcerans headspace samples were compared with four from sterile growth medium and laboratory environmental air. The univariate analysis resulted in the selection of 24 retained VOC fragments and a perfect differentiation between cultures and controls. Sixteen of 24 fragments were identified, resulting in eleven unique compounds, mainly alkanes. Methylcyclohexane was the best performing compound. Based on these 24 fragments, headspace samples originating from gauzes of 50 open skin lesions (12 qPCR positive and 38 negative) were analysed and an AUC of 0.740 (95%-CI 0.583–0.897) was obtained. As this is an experimental study, future research has to confirm whether the identified compounds can serve as novel biomarkers. Author summary: In this study the odour of bacteria that cause Buruli ulcer was examined. First, the air of bacteria cultured in the lab was analysed and compared with controls. Next, air was sampled from gauzes worn by people in the Kongo Central province, the Democratic Republic of Congo. Their lesions were compatible with Buruli ulcer disease but only 12 out of 50 were infected with the Buruli bacterium. In the odour of the Buruli bacteria and in gauzes from people with Buruli ulcer, several chemicals were present more abundantly than in gauzes from people with wounds caused by other diseases. Future research has to determine whether these chemicals can become part of a new test for Buruli ulcer disease. [ABSTRACT FROM AUTHOR]