The rapid rise in drug-resistant tuberculosis poses a serious threat to public health and demands the discovery of new anti-mycobacterial agents. Medicinal plants are a proven potential source of bioactive compounds; however, identifying those responsible for the putative anti-mycobacterial action still remains a challenging task. In this study, we undertook a systematic network pharmacology approach to identify and evaluate anti-mycobacterial compounds from a traditional plant, Acacia nilotica, as a model system. The protein-protein interaction network revealed 17 key pathways in M. tuberculosis encompassing 40 unique druggable targets that are necessary for its growth and survival. The phytochemicals of A. nilotica were preferentially found to interfere with the cell division and cell wall biogenesis proteins, especially FtsZ and Mur. Notably, the compounds epigallocatechin, ellagic acid, chlorogenic acid, and D-pinitol were found to exhibit a potential polypharmacological effect against multiple proteins. Further, in vitro studies confirmed that the selected candidates, chlorogenic acid, and ellagic acid exhibited potent anti-mycobacterial activity (against M. smegmatis) with specific inhibition of purified M.tb FtsZ enzyme. Taken together, the present study demonstrates that network pharmacology combined with molecular docking can be utilized as an efficient approach to identify potential bioactive phytochemicals from natural products along with their mechanism of action. Hence, the compounds identified in this study can be potential lead candidates for developing novel anti-mycobacterial drugs, while the key proteins identified here can be potential drug targets.