The recent increase in the incidence of tuberculosis (TB) with the emergence of multidrug-resistant (MDR) cases has lead to the search for new TB-drugs. Mycobacterium tuberculosis is a complex, resilient organism, and it is important to note that new drugs are required which can reduce TB’s six month treatment time and can be effective against drug-resistant strains of mycobacteria. Plants contain numerous biological active compounds, many of which have been shown to have antimicrobial activity. The search for biologically active extracts based on traditional use of plants is relevant due to the appearance of microbial resistance to many antibiotics and the occurrence of fatal opportunistic infections. Ethanol extracts of seven selected ethnobotanically South African medicinal plants (Artemisia afra, Dodonaea angustifolia, Drosera capensis, Galenia africana, Prunus africana, Syzygium cordatum and Ziziphus mucronata) were investigated for their antimycobacterial activity against two Mycobacterium species. The minimum inhibitory concentration (MIC) of ethanol extracts of A. afra, Dodonaea angustifolia, Drosera capensis and G. africana ranged from 0.781 to 6.25 mg/mL against a non-pathogenic strain of mycobacteria, ‘M. smegmatis’. G. africana showed the best activity, exhibiting an MIC of 0.781 mg/mL and a minimum bactericidal concentration (MBC) of 1.563 mg/mL against M. smegmatis. A drug sensitive strain of M. tuberculosis was found to be susceptible to the ethanol extracts of Dodonaea angustifolia and G. africana. (MICs 5.0 and 1.2 mg/mL respectively) when using the rapid radiometric-BACTEC method. The phytochemical analysis of G. africana led to the isolation and identification of three known compounds namely; (2S)-5,7,2'-trihydroxyflavanone, (E)-3,2',4'-trihydroxychalcone (not reported from natural sources) and (E)-2',4'-dihydroxychalcone. A novel chalcone ‘(E)-3,2',4'-trihydroxy-3'-methoxychalcone’ was also isolated from the ethanol extract of G. africana. Isolation of (2S)-5,7,2'-trihydroxyflavanone, (E)-3,2',4'-trihydroxychalcone and E)-3,2',4'-trihydroxy-3'-methoxychalcone was reported for the first time from this plant. The MIC of novel compound against M. tuberculosis was found to be 50.0 µg/mL whereas (2S)-5,7,2'-trihydroxyflavanone and (E)-3,2',4'-trihydroxychalcone exhibited an MIC of 100.0 µg/mL. During synergistic studies using (2S)-5,7,2'-trihydroxyflavanone and (E)-2',4'-dihydroxychalcone with the antituberculosis drug INH, it was found that the MICs of INH and the compounds were reduced sixteen and eight-fold respectively, resulting in a Fractional Inhibitory Concentration (FIC) of 0.1250 and 0.1875 respectively. The synergistic effect of two isolated compounds (2S)-5,7,2'-trihydroxyflavanone and (E)-2',4'-dihydroxychalcone) in in vitro studies also showed synergistic action, reducing their original MICs four-fold resulting in a FIC of 0.5. Since (2S)-5,7,2'-trihydroxyflavanone and (E)-2',4'-dihydroxychalcone from G. africana showed synergistic activity with INH, it is speculated that the compounds might have similar mechanism as that of INH. However, mechanistic studies on these compounds should be done in order to get an indication of the ‘flavonoids and chalcones’ interferences on mycolic acid synthesis, membrane synthesis and enzyme inhibition. Our investigation on the NADPH oxidase activity of (2S)-5,7,2'-trihydroxyflavanone with Mtr, found that this compound failed to exhibit any NADPH oxidase activity at 800 µM concentrations. Mtr is evidently not the target for the antimycobacterial activity of (2S)-5,7,2'-trihydroxyflavanone. Fifty percent inhibitory concentration of the ethanol extract of G. africana, and the two purified compounds, (2S)-5,7,2'-trihydroxyflavanone and (E)-2',4'-dihydroxychalcone were found to be 120.0; 110.3 and 80.2 µg/mL respectively against the U937 cells. The MIC of the ethanol extract of G. africana in U937 macrophages infected with M. tuberculosis was found to be 50.0 µg/mL indicating the extract’s intracellular antimycobacterial activity in real physiological conditions. The two purified compounds also showed good intracellular antimycobacterial activity. The MICs of (2S)-5,7,2'-trihydroxyflavanone and (E)-2',4'-dihydroxychalcone were found to be 100 and 50 µg/mL respectively. This study indicated that the intracellular activity of the ethanol extract is significant in macrophages. The activity might be due to M. tuberculosis being unable to avoid macrophage killing and its survival during phagocytosis, (including inhibition of phagosome-lysosome fusion, inhibition of the acidification of phagosomes, resistance to killing by reactive oxygen intermediates and modification of the lipid composition of the mycobacterial cell membrane, thereby altering its capacity to interact with immune or inflammatory cells). It can be concluded that the traditional use of G. africana for TB has been scientifically validated to some extent. Isolated compounds and the ethanol extract of the plant warrant further investigation for their potential as antimycobacterial agents. Since synergistic activity of purified compounds with existing antituberculosis drug INH, was significant, it will be worthwhile evaluating the efficacy of purified compounds in combination with TB-drugs in pre-clinical studies.