Chitosan nanoparticles (CH NPs), ZrO2, Ni/ZrO2. CH/ZrO2 and CH/Ni/ZrO2 complex have been synthesized then its antibacterial mechanisms of nanomaterials against Escherichia coli (E. coli) was evaluated by agar well diffusion and systematically analyzed by measuring the diameter of the inhibition zone. X-ray diffraction (XRD) patterns and Fourier transform infrared (FTIR) spectra and Energy dispersive X-ray (EDX) results demonstrated the formation of CH/Ni/ZrO2 matrix. Also, the crystalline sizes were found to be 15, 13, 10 and 8 nm for ZrO2, Ni/ZrO2, CH/ZrO2 and CH/Ni/ZrO2 respectively. The FTIR spectrum revealed bands ranging from 400 to 800 cm−1, indicating the presence of metal–oxygen bonds. Additionally, bands at 1620, 1523, and 1300 cm−1 suggested the presence of amide groups, confirming the presence of CH in the produced CH/Ni/ZrO2 nanocomposite. In SEM analysis, the irregular spherical nanoparticles are observed in case ZrO2 and CH/ZrO2 samples, while spherical with an oval-shaped structure are observed in case of Ni/ZrO2 and CH/Ni/ZrO2 sample. Mean diameter of inhibition zone were observed to be 13 ± 2.51, 12 ± 2.08, 16 ± 1.3 mm,17 ± 2.3 mm and 18 ± 0.8 nm for CH, ZrO2, Ni/ZrO2, CH/ZrO2 and CH/Ni/ZrO2 respectively. The eco-friendly and cost-effective synthesis of Chitosan assisted Ni/ZrO2 NPs can be utilized as an effective antibacterial agent.