Stevia a "Latin American herb‟ is the world‟s only natural sweetener with zero calories, zero carbohydrates and a zero glycemic index. In the present investigation, the in vitro regeneration of Stevia rebaudiana was performed through callogenesis and organogenesis from different explants (Leaves, Inter node, Shoot discs). Leaves explants showed best callus induction response when cultured on MS + (2.0 µM BAP + 1.0 µM NAA). However, the best callus initiation from leaf explants was obtained on MS medium supplemented with 1.0 µM NAA + 1.0 µM Kn. Optimization results of medium type and carbon source confirm 1X MS medium and glucose (3%) respectively best for callus initiation. Shoot initiation was achieved on 1X MS medium supplemented with 5.0 µM BAP + 1.0 µM NAA. In vitro raised shoots of Stevia showed best rooting on 1X MS + (1.0 µM IAA). The methanolic extracts of regenerated plants and callus culture of Stevia showed best antibacterial and antifungal activity against a number of microorganisms. Antiprotozoal activity of methanolic extract was also tested and found satisfactory. In vitro regeneration protocol of Stevia through callus culture is advantageous for enhanced multiplication of superior Stevia cultivars. Furthermore, callus from leaf explants can be a good source for production of antimicrobial and antiprotozoal compound of Stevia through bioreactor.