Recent investigations have demonstrated that nickel ferrite nanoparticles and their derivatives have toxicity effects on bacterial cells. In this study, we have prepared nickel ferrite nanoparticles (Ni/NiFe2O4) and nickel/nickel ferrite graphene oxide (Ni/NiFe2O4-GO) nanocomposite and evaluated their toxic effects on E. coli cells ATCC 25922. The prepared nanomaterials were characterized using X-ray diffraction, Raman spectroscopy, scanning electron microscopy, transmission electron microscopy, and vibrating sample magnetometry techniques. The toxicity was evaluated using variations in cell viability, cell morphology, protein degradation, and oxidative stress. Ni/NiFe2O4-GO nanocomposites likewise prompt oxidative stress proved by the age of reactive oxygen species (ROS) and exhaustion of antioxidant glutathione. This is the first report indicating that Ni/NiFe2O4-GO nanocomposite-initiated cell death in E. coli through ROS age and oxidative stress.