BackgroundZanha africana (Radlk.) Exell extracts are commonly utilized in Tanzania and other countries for treating conditions associated with HIV and AIDS, including tuberculosis (TB). In Tanzania, it is listed among the most frequently traded plant species for medicinal use in different marketplaces. However, limited literature exists regarding its antimycobacterial properties and safety profile. ObjectiveThis study aimed to evaluate Z. africana ethanolic extract for safety and antimycobacterial activity. MethodsThe antimycobacterial activity of the crude extracts was assessed using the broth microdilution method against various non-pathogenic mycobacteria strains including Mycobacterium aurum (MA), Mycobacterium madagascariense (MM), and Mycobacterium indicus pranii (MIP). Bioassay-guided fractionation was employed to isolate the active compounds. The safety profile of the crude extract was investigated through cytotoxicity assay on peripheral blood mononuclear cells (PBMCs) and the brine shrimp lethality test. ResultsThe 80% ethanolic extracts of Z. africana demonstrated activity against all tested non-pathogenic mycobacteria. The antimycobacterial activity of ethanolic extract of the stem bark was 625 µg/mL, 313 µg/mL and 125 µg/mL against MA, MM, and MIP, respectively. The most potent fraction was the ethyl acetate fraction, from which two active compounds were isolated: proanthocyanidin A2; Compound (1) and its monomer epicatechin, Compound (2). Compound (1) exhibited higher antimycobacterial activity compared to compound (2) with minimum inhibitory concentrations (MICs) of 19.50 µg/mL and 12.98 µg/mL against MM and MIP respectively. The ethanolic extract did not demonstrate toxicity on PBMCs and brine shrimps. ConclusionThis study demonstrates that Z. africana extracts and the isolated compounds possess antimycobacterial activity. This provides evidence supporting the traditional use of Z. africana extracts for TB management. Further research is recommended to comprehensively evaluate the efficacy and safety of the plant constituents using different models.