SBA-16 is a mesoporous nanostructure with high surface area and cubic symmetry which make them an ideal carrier to carry different nanoparticles. Zinc peroxide (ZnO₂) is a novel nanoparticle that inhibits the growth of microorganisms due to its antiseptic property. Thin film of SBA-16 and SBA-16/ZnO₂ was deposited on glass slide by spin coating method. Anti-biofilm property of the thin films was examined against E. coli and S. aureus bacteria. Structural property was found out by wide angle X-ray diffraction (XRD), small angle X-ray scattering (SAXS) and High Resolution Transmission Electron Microscope (HRTEM). Fourier Transform Infrared Spectrophotometer (FTIR) was used to confirmation the presence of ZnO₂ in Nano-composite and AFM utilized for surface topography of thin films. Fluorescent microscope image clearly revealed that SBA-16/ZnO₂ nanocomposite thin film significantly inhibit the biofilm formation against both E. coli (gram negative bacteria) as well as S. aureus (gram positive bacteria) in comparision to SBA-16. The antimicrobial behavior of ZnO nanoparticle is due to formation of H₂O₂ as reactive oxygen species (ROS) to which the bacterial cells are permeable. H₂O₂ is a powerful oxidizing agents that oxidized the mycobacterial elements like DNA and proteins which results into the toxicity of the bacteria cell.