Herein we report synergy in antimicrobial activity of a chitosan silver nanopartiele (CS-Ag NP) composite in the presence of molecular iodine. Green fluorescent protein (GFP) expressing recombinant Escherichia call bacteria have been used to test the efficacy and establish the mechanism of action. Experimental evidence indicate significantly high bactericidal activity of the nanocomposite in the presence of iodine than either due to the composite, chitosan, Ag NP or iodine only. Transmission electron microscopy measurements revealed at of bacteria to the composite. In addition, flow cytometry results supported definite occurrence of cell wall damage of the bacteria treated with the composite in the presence of iodine. Further, the nanocomposite and iodine combination was found to exert reactive oxygen species ( ROS) generated oxidative stress in the cytoplasm of bacterial cells, leading to cell death. Elucidation of die mechanism of synergy due to three potential antibacterial components suggested that on the surface of Ag NPs molecular iodine possibly generated iodine atom thus contributing toward free radical induced oxidative stress, whereas chitosan and Ag NPs facilitated the process of cell killing and thus collectively enhanced the potency of antimicrobial effect at the lowest concentrations of individual components.