Hybrid nano-supra molecular structured materials can boost the functionality of nano- or supra-molecular materials by providing increased reactivity and conductivity, or by simply improving their mechanical stability. Herein, the studies in materials science exploring hybrid systems are investigated from the perspective of two important related applications: healthcare and food safety. Interfacing phase strategy was applied, and ZnAl layered double hydroxide-chitosan hybrids, prepared by the urea method (U-LDH/CS), were successfully synthesized under the conditions of different chitosan(CS) concentrations with a Zn/Al molar ratio of 5.0. The structure and surface properties of the U-LDH/CS hybrids were characterized by X-ray diffraction (XRD), Fourier-transform infrared spectrometer(FTIR), scanning electronmicroscopy (SEM), ultravioletvisible (UV-Vis), and zero point charge (ZPC) techniques, where the effect of CS concentration on the structure and surface properties was investigated. The use of the U-LDH/CS hybrids as antimicrobial agents against Escherichia coli, Staphylococcus aureus, and Penicillium cyclopium was investigated in order to clarify the relationship between microstructure and antimicrobial ability. The hybrid prepared in a CS concentration of 1.0 g∙L−1 (U-LDH/CS1) exhibited the best antimicrobial activity and exhibited average inhibition zones of 24.2, 30.4, and 22.3mm against Escherichia coli, Staphylococcus aureus, and Penicillium cyclopium, respectively. The results showed that the appropriate addition of CS molecules could increase antimicrobial ability against microorganisms.