Antibacterial efficacy of silver nanoparticles (Ag NPs) deposited alternatively layer by layer (LBL) on chitosan polymer in the form of a thin film over a quartz plate and stainless steel strip has been studied. An eight-bilayer chitosan/silver (Cs/Ag)8 hybrid was prepared having a known concentration of silver. Techniques such as UV-visible spectroscopy, inductively coupled plasma optical emission spectroscopy (ICP-OES), and atomic force microscopy (AFM) were carried out to understand and elucidate the physical nature of the film. Gram-negative bacteria, Escherichia coli (E. coli), were used as a test sample in saline solution for antibacterial studies. The growth inhibition at different intervals of contact time and, more importantly, the antibacterial properties of the hybrid film on repeated cycling in saline solution have been demonstrated. AFM studies are carried out for the first time on the microbe to know the morphological changes affected by the hybrid film. The hybrid films on aging (3 months) are found to be as bioactive as before. Cytotoxicity experiments indicated good biocompatibility. The hybrid can be a promising bioactive material for the prevention of biofilms specific to E. coli and in purification of water for safe drinking.