The essential oil of Vetiveria zizanioides showed significant antimycobacterial activity against the drug-resistant strains of Mycobacterium smegmatis, which on activity guided fractionation afforded four bioactive fractions Vz-2, Vz-7, Vz-8, and Vz-9. Further purification of these bioactive fractions over preparative thin layer chromatographic resulted in the characterization of six compounds: 5, 10-pentadecadiyn-1-ol (1), a-curcumene (3), hydroxy junipene (4), (?) cycloisosativene (5), valencine (6), and selino 3,7 (11)-diene (7). All these compounds showed significant antimycobacterial activity against the drug-resistant strains (MDR-R and MDR-40) of M. smegmatis and their MIC was in the range of 31.25–62.5 lg/ml. These results may be of great help in antimycobacterial drug development from a very common, inexpensive, and non toxic natural product.