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The menace of antimicrobial resistance continues to increase and hence the need to discover new antibiotics, especially alternative and effective sources such as hybrid organic-inorganic, organic-organic materials, and other combinations. In this study, an antimicrobial hybrid supra-nano material was prepared by the bi-titration synthesis method of chitosan (CS) and ZnAl layered double hydroxide. Fourier-transform infrared spectrometer (FTIR), thermogravimetric and differential thermal gravimetric (TGA/DTG), ultraviolet-visible (UV-Vis), X-ray diffraction (XRD), and scanning electron microscopy (SEM) analyses indicated that the ZnAl/CS hybrid exhibited low crystallinity with high thermal stability. The results of ZnAl/CS characterization showed the characteristic properties of the individual components ZnAl and CS, indicating a successful preparation of the ZnAl/CS hybrid. The antibacterial tests revealed that the ZnAl/CS hybrid possessed an enhanced antimicrobial effect against both Escherichia coli (E. coli, MTCC 739) and Penicilliumcyclopium (P. cyclopium, AS 3.4513). Under the central composite design (CCD) of the response surface methodology (RSM) tool, the parameters of the hybrid synthesis reaction were optimized and the result obtained was as follows: reaction pH was 11.3, reagent Zn/Al ratio was 3.27, and chitosan concentration was 1.07 g/L. After optimization, it was found that the antibacterial activity of ZnAl/CS was strengthened against E. coli as evidenced by a widening of the inhibition zone of about 41.6%. The antibacterial activity of ZnAl/CS was mainly due to the reactivation of the antibacterial activity of CS associated with the release of Zn2+ and Al3+ metal ions in addition to ZnO, Al2O3, and ZnAl2O4 compounds resulting from the method of preparation. [ABSTRACT FROM AUTHOR]