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Buruli ulcer is a devastating skin infection caused by Mycobacterium ulcerans. It is endemic in the Amansie West district of the Ashanti region of Ghana where patients rely mainly on herbal preparations from practitioners. This work aimed at validating some preparations employed by traditional healers in the Amansie West district for the management of Buruli ulcer. Four herbal preparations, obtained from practitioners in four communities were tested against the clinical M. ulcerans strain (NM203) using the resazurin microplate assay. Cytotoxicity test was done on the extracts using Chang liver cells. Chemical profiling of the active formulation was done using High-performance liquid chromatography. Some traditional herbal practitioners considered painless wounds in the extremities as diagnostic of BU whereas others relied on diagnosis of BU from a recognized conventional healthcare facility before proceeding to treat with herbal products. A formulation containing the roots of Cryptolepis sanguinolenta and seeds of Picralima nitida, as plant ingredients, was considerably active (MIC = 32µg/mL). The preparations were non-cytotoxic to Chang- liver cells. The herbal preparations showed considerable antimycobacterium ulcerans activity and are not toxic. A major peak in the HPLC chromatogram of the active formulation with a retention time of 17.86 min was identified as a marker for the quality analytics of the active formulation. A traditional herbal preparation used in Buruli ulcer endemic community was validated to show antimycobacterium ulcerans activity. Validation of similar preparations could afford novel drugs or products for this neglected tropical disease.