A convenient and faster protocol established for clonal propagation of Stevia rebaudiana Bertoni through callus and shoot culture, with its antimicrobial screening. MS medium with (0.5-2.0mg/L) BAP was found to be optimal for shoot initiation. A high-frequency multiplication rate was obtained from nodal segments in MS medium supplemented with 0.5 mg/l BAP+2.0 mg/l Kn. Root induction occurred with IBA 1.0 mg/l then transferred to poly tray containing sand, vermiculite and soil (1:1:2) and acclimatized for a period of 3 weeks. In vivo grown leaf extracts in different solvent system exhibited a concentration dependent antibacterial and antifungal inhibition against standard by disc diffusion method. The methanolic extracts of Stevia leaf showed best antibacterial and antifungal activity against a number of microorganisms. This proved Stevia’s potential as antimicrobial agent from non antibiotics sources. Therefore, commercial manufacture of active constituents from these improved elite lines would be useful and profitable.