Justicia adhatoda L. is an Indian medicinal plant traditionally used to treat respiratory ailments in Ayurvedic and Unani medicines. It is widespread in the Indian subcontinent. The plant leaves are known to have broad range of pharmacological activities including analgesic, anti-inflammatory, antispasmodic and antibacterial properties. Some studies have highlighted the antimicrobial effects of its major bioactive components like vasicine and vasicinone However, not much work has been done to explore the enormous benefits that the bioactive components might hold. Hence, in the present study, we explored the plant for its potential as an effective anti- mycobacterial agent. We aim to investigate the antimycobacterial effect of J. adhatoda L. plant leaf extracts on Mycobacterium smegmatis and Mycobacterium bovis (BCG) and identify, and isolate th bioactive component(s) for the plant leaf extract. The isolated components were also analyzed further for their antimycobacterial activity in synergy with Isoniazid. The leaves of J. adhatoda L. were powdered and extracted with ethanol, water, ethyl acetate, and hexane and antimycobacterial activity was assessed by MABA. The ethanol extract showed >96% and 98% reduction in colony-forming units (CFU) at 100µg/ml on Mycobacterium smegmatis and Mycobacterium bovis (BCG) respectively. Active phytoconstituent from ethanol extract was isolated and further fractionated via Prep-TLC. They were bioassayed against M. smegmatis and M. bovis (BCG) to study cytotoxicity, synergy, and external damage on the bacteria. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) were carried out on treated bacilli to observe the external and internal cellular damage. The isolated fractions showed strong anti-mycobacterial activity, with low cytotoxicity and synergism when used with isoniazid. When subjected to electron microscopy, the fractions were found to adversely affect the cell wall and membrane of BCG, causing cytoplasmic leakage. Our study demonstrated that the active fractions isolated from J. adhatoda L. could be evaluated further for potentially effective anti-mycobacterial agents.