Vernonia adoensis is a folk herbal medicine traditionally used to treat tuberculosis and tuberculosis-related ailments. The study aimed to determine the antitubercular activity of V. adoensis and its possible mode of action against Mycobacterium smegmatis. Extracts and phytochemicals were tested for growth inhibitory and bactericidal activities against M. smegmatis. The effects of the most effective plant extract on drug transport across the membrane and its ability to cause membrane and protein leakage in mycobacterial cells were determined. Its capacity to quench DPPH (2, 2-diphenyl-1-picryl-hydrazyl) was also evaluated. The ethyl acetate extract from Vernonia adoensis leaf was most effective against M. smegmatis with a minimum inhibitory concentration and minimum bactericidal concentration of 63 μg/ml and 125 μg/ml respectively. The most active phytochemical was the alkaloid fraction with an MIC of 125 μg/ml. Significant nucleic acid and protein leakage in M. smegmatis was observed after exposure to the leaf extract. The extract did not possess significant free radical scavenging activity and did not affect drug transport in mycobacterial cells. Results showed that the ethyl acetate leaf extract exhibited potential antimycobacterial activity against M. smegmatis. Cell membrane disruption resulting in protein and nucleic acid leakage could be the plant's possible mode of action.