The roots of Euclea natalensis are used by the indigenous people of southern Africa against various bacterial infections. Six naphthoquinones; diospyrin, isodiospyrin, mamegakinone, 7-methyljuglone, neodiospyrin and shinanolone were isolated from root extracts of this plant. In the case of neodiospyrin, this was a first time isolate. The activity of the six isolated compounds were tested against Mycobacterium tuberculosis using the radiometric BACTEC assay, providing a first report on the antimycobacterial activity of isodiospyrin, neodiospyrin and mamegakinone. The MIC values of diospyrin (8.0 μg/ml), isodiospyrin (10.0 μg/ml), 7-methyljuglone (0.5 μg/ml) and neodiospyrin (10.0 μg/ml) compared well to those of the known antimycobacterial drugs, ethambutol, isoniazid and rifampicin. A hypothesis on the structure-activity relationship for the naphthoquinones and a possible mode of action is discussed.