Background: Brucellosis is one of the most prevalent bacterial zoonosis which is transmitted to humans from animals. As an alternative to conventional antibiotics, medicinal plants are valuable resources for new agents against antibiotic-resistant strains. Objective: To evaluate the antibrucellosis activity of different medicinal plants collected from the Western Ghats against Brucella abortus, Brucella melitensis, Brucella suis. Identification and characterization of the bioactive metabolites of the potent antibrucellosis agent by Thin Layer Chromatography and Gas chromatography mass spectroscopy. Methods: Antibacterial assay was carried for the ethanolic extract of different medicinal plants, the potential and effective medicinal plants extract was subjected for purification by TLC and the bioactive metabolites were characterized by the GC MS analysis. Results: Acacia nelotica, Terminalia arjuna, Eugenia jambolana and Callistemon citrinus showed the antibrucellosis activity comparatively Callistemon citrinus had the strong antibrucellosis activity. Further the crude sample was purified by TLC profiling, compounds with different retention factor were screened for antibrucellosis activity, and the bioactive metabolites were identified by GC-MS analysis. Conclusion: For the first time the different medicinal plants from Western Ghats were screened for the antibrucellosis activity. The crude and TLC purified Callistemon citrinus ethanolic extract exhibited strong antibrucellosis activity. The bioactive compounds identified were reported for the first time and the bioactive metabolites identified exhibited as potential antibacterial agents against brucellosis and other Human pathogens.