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A novel nanomaterial Bacitracin-Ag Nanoclusters (Bacitracin-AgNCs) was formed to achieve a better antibacterial effect on Shigella flexneri which poses a serious threat to human health. In the current study, X-ray photoelectron spectrometer (XPS), Fourier transform infrared (FTIR), field-emission scanning electron microscopy (FESEM), high resolution transmission electron microscopy (HR-TEM) and thermal gravimetric analysis (TGA) were used to characterize the properties of composited Bacitracin-AgNCs. Furthermore, the inhibitory effects of Bacitracin-AgNCs against S. flexneri were explored, and the inhibition mechanism was discussed in terms of its aspects of cell membrane ravage, ATPase activity decline and biofilm inhibition. The results reveal that the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Bacitracin-AgNCs against S. flexneri were 0.03 mg/mL and 4 mg/mL. Bacitracin-AgNCs may cause irreversible impairment to cells and greatly change the cell morphology. The cell membrane integrity of S. flexneri was destroyed with changes in the characteristics of membrane permeability and intracellular substances leakage. Moreover, our study further proved that Bacitracin-AgNCs significantly inhibited the formation of S. flexneri biofilms and reduced the number of viable bacteria in biofilm. These findings provide a potential method for the exploitation of organic composite nanomaterials as a novel antimicrobial agent and its application in the food industry. [ABSTRACT FROM AUTHOR]