Biological synthesis involves the reduction and stabilization of metal ions using different types of organisms. Plants are an attractive source for silver nanoparticles (Ag NPs) synthesis because of their strong reducing potentials by production of a wide range of secondary metabolites. The present study describes the biosynthesis of Ag NPs using plant extract of Ocimum basilicum extract grown under in vitro condition for the first time. The surface Plasmon resonance found at 450 nm confirmed the formation of Ag NPs. FESEM images revealed relatively spherical- shaped of Ag NPs. The biosynthesized Ag NPs were crystalline in nature with mean diameter about 13.82 nm. FTIR results expounded the functional groups of plant extract responsible for the bio-reduction of silver ions and the interactions between them. The obtained nanoparticles showed good inhibitory activity against both Gram positive and Gram negative bacteria. These results suggest that with changes in plant's culture condition it may be possible to obtain nanoparticles with desired characteristics.