Recently, magnetic nanoparticles have been considered to bacterial removal, due to monodisperse size, the ability of functionalization or modification. A novel amino-terminated poly (amidoamine) generation 5 (PAMAM-G5) functionalized Fe3O4/SiO2- GPTMS magnetic nanomaterial with a core–shell structure was developed. The nanocomposite was employed to determine antibacterial properties using paper disc diffusion, broth microdilution (Minimum Inhibitory Concentration-MIC and Minimum Bactericidal Concentration -MBC determination), adsorption, and surface plate count (Colony Forming Unit-CFU/ml) methods. The nanocomposite was characterized by FTIR, N2 adsorption/desorption, Zeta potential, FE-SEM, TEM, EDX, DLS, XRD, TGA and VSM techniques. The impacts of different parameters including primary bacteria count, solution pH, reaction time and the nanocomposite dosage were studied. The nanocomposite cytotoxicity was investigated on AGS cell line by MTT assay. Nanocomposite was effective in preventing both bacterial strains growth. The MIC and MBC values for E. coli and S. aureus were determined 4 and 8 μg/ml and 16 μg/ml for both strains respectively. Synthesized nanocomposite as antibacterial in an aqueous solution, was elimination effectiveness of 100% for both selected bacteria in optimum condition (pH = 7, nanocomposite dosage = 16 μg/ml, run time = 60 min, and initial bacteria count = 103 CFU/ml). FE-SEM analysis revealed morphological variations and the mechanism of killing and trapping the bacteria by nanocomposite. The nanocomposite cytotoxicity on the cell indicated the impact of exposure time and nanocomposite concentration. The reduction in cell viability in the concentration of 16 μg/ml after 48 and 72 h treatment was shown to be 27.4 and 28.7%, respectively. The nanocomposite is able to reduce the E. coli and S. aureus count in drinking water, which meets the drinking water standards according to WHO guidelines.