The occurrence of bacterial infection has increased significantly over last few decades. Thus, it has arisen concern of research to produce new effective drugs to control bacterial infection. The new approach based on green synthesized nanoparticles (NPs) will be a turning point for addressing growing global resistance of antibiotics. In this study, the green synthesis approach was used to synthesize Cadmium Sulfide nanoparticles by utilizing Sesamum radiatum leaves extract. The prepared Cadmium Sulfide nanoparticles were characterized by UV-Vis spectrophotometer, Fourier Transformation infrared (FTIR), Scanning Electron Microscope (SEM) and Thermal gravimetric analysis (TGA). The UV-Vis spectra of CdSNPs revealed abroad absorption band between 300 nm - 350 nm contributing to the surface Plasmon Vibration of metal nanoparticles. The presence of functional groups of amines, aliphatic amines, alkenes, alkynes, aryl, alcohols, carboxylic acid, alkyl halides, aromatics, nitro group, phenol, ester and anhydrides were confirmed by FTIR analysis. SEM obtained the information regarding to the surface morphology. TGA analysis was carried out to prove that the stability of CdSNPs. The antibacterial activity of CdSNPs against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was carried out by discs diffusion method with increasing concentration (10mg/ml-30mg/ml). The increasing concentration of nanoparticles enchanted inhibition zone. The DNA cleavage activity was performed to visualize the ability of the CdSNPs to cleave pBR322 DNA which indicates that the disappear of band I from the circular plasmid DNA using gel electrophoresis.