The plant mediated synthesis of metallic nanoparticles is preferred over chemical and physical methods owing to their easy synthesis, simplicity, eco-friendliness, stability and developed as lucrative field of green nanotechnology. Use of calli in place of other plant organ avoids the mass collection of plant from their wild habitat and offers the synthesis of nanoparticles for all those plants which are critically endangered or are not available throughout the year. In the present study, an alternative and novel protocol has been developed for the biosynthesis of Zinc Oxide nanoparticles (ZnO NPs) using in vitro culture technique of plant tissue culture as a basic platform for the plant material. Thus the present work aimed to synthesized ZnO NPs using the calli of Viola canescens (Banafsha) and study anti-microbial activity of synthesized nanoparticles. The calli were generated from the leaf and petiole explants, using the auxin (2,4-D and NAA) and cytokinin (Kn). Interaction of callus extract with Zinc nitrate hexahydarate extract for the bio-reduction of Zinc nitrate hexahydarate to its nanoparticles showed potency of calli to reduce Zinc nitrate to its nanosizes. The confirmation for the synthesis of the nanoparticles was done by UV-visible analysis followed by characterization of synthesized nanoparticles with XRD, FTIR and SEM techniques. X-ray diffraction analysis showed that nanoparticles exhibited the crystalline nature with wurtzite hexagonal shape and average size was less than 09 nm. FTIR revealed the presence of biomolecules which act as reduction and capping agent for the synthesis of ZnO NPs. Antibacterial activity of the synthesized nanoparticles against four different pathogenic (gram positive and gram negative) strains showed clear zone of inhibitions that confirm the potency of nanoparticles as an antibacterial agent. The present work explored the potential of the callus as bio-reduction material for nanoparticles synthesis.