Zinc (Zn) and, alternatively, nickel (Ni) substituted cobalt ferrite (CF) nanoparticles (NPs) were prepared by sol-gel method. X-ray diffraction analysis revealed the formation of cubic structure of cobalt ferrite. FTIR analysis confirmed the vibrational band located at 550-580 cm that belongs to the M - O bond (M = Ni, and Zn). The alteration of the surface morphology of CF after the addition of Zn and Ni ions was observed from scanning electron microscopic images. The additional peaks in the energy dispersive X-ray diffraction (EDX) analysis spectra were found to correspond to Zn and Ni. The presence of Zn and, alternatively, Ni ions enhanced the biocidal properties of CF NPs against gram negative organisms, in a concentration and time-dependent manner. Furthermore, exposure to CF, CF-Zn and CF-Ni NPs decreased metabolic activity due to the damage of extra polymorphic substances, live/dead cell variation, architecture and surface integrity of the cells. Altogether, the present investigation provides the basis of metal ion substituted metal oxide NPs as anti-biofilm agents against gram-positive and gram-negative bacteria.