The discovery of antimalarial artemisinin from Artemisia annua L. is an example of how Traditional Chinese Medicine (TCM) may be exploited to meet a recognized need. In this study, we systemically investigated A. annua L. for its antimicrobial activity and assessed it as a source of bioactive natural products for anti-mycobacterial activity. We used a silica gel column to perform antimicrobial activity-guided purification of the A. annua leaf, whose identity was confirmed by rbcL DNA barcoding, and used UHPLC-HRMS and NMR to elucidate the structure of purified active compounds. The antimicrobial activity of crude extracts, isolated compounds and the control artemisinin (Apollo Scientific Ltd) was assessed against Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus , methicillin-resistant Staphylococcus aureus (MRSA), Mycobacterium smegmatis strains by serial micro dilution method (31.25-1000 μg/mL). The isolated compounds were tested for synergistic effects against mycobacterium. Bioactive compounds were purified and identified as deoxyartemisinin and artemisinic acid. Artemisinic acid (MIC 250 μg/mL) was more effective in comparison to deoxyartemisinin (MIC 500 μg/mL) and artemisinin (MIC 1000 μg/mL) against M. smegmatis . We used a molecular docking approach to investigate the interactions between selected anti-mycobacterial compounds and proteins involved in vital physiological functions in M. tuberculosis , namely MtPks13 , MtPknB , MtPanK , MtKasA , MtInhA and MtDprE1 and found artemisinic acid showed docking scores superior to the control inhibiters for MtKasA , suggesting it to be a potential nick for further in vitro biological evaluation and anti-TB drug design. ### Competing Interest Statement The authors have declared no competing interest.

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