Sesamum radiatum leaves are widely consumed as food supplement and for ethno-medicinal purposes and S. radiatum use in many researches. This study aimed to characterize the newly prepared Silver nanoparticle using S. radiatum and to evaluate the antibacterial activity and DNA cleavage ability of Silver nanoparticles. Silver nanoparticles are one of the metal nanoparticles which have varied and different properties when compared to other material. Silver nanoparticles are synthesized by the chemical reduction of silver nitrate. These silver nanoparticles are characterized by the different techniques like UV-Vis spectroscopy for the structural characterization, SEM to know the structural morphology and composition, FT-IR to know how many samples was absorb or emission and TGA to examining the mass changes of a sample as a function off temperature. Antibacterial activity of AgNPs against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was carried out by disc diffusion method of nanoparticle enhance inhibition zone was observed. The interaction of DNA bases and corresponding nucleotides with silver nanoparticles (AgNPs) bio functionalized by DNA pBR322 has been investigated by absorption spectroscopy and dynamic light scattering.