In this work, a novel chitosan Schiff base 4-(2-Hydroxyaniline)pent-3-en-2-one chitosan (2-HyA-CS) and its ZnO nanocomposite (2-HyA-CS/ZnO) were sensitized and characterized by appropriate methods; FTIR, XRD, Elemental analysis, SEM, TEM and TGA. The result of characterization methods confirms the preparation of 2-HyA-CS and 2-HyA-CS/ZnO. The SEM images reveal that chitosan, 2-HyA-CS, and 2-HyA-CS/ZnO have a varied roughness and porous surfaces. The reason for this difference was attributed to the formation of Schiff base 2-HyA-CS and the presence of ZnO nanoparticles in 2-HyA-CS/ZnO. The patterns of XRD and FTIR confirm the formation of 2-HyA-CS and 2-HyA-CS/ZnO. The degree of substitution (DS) of modified chitosan 2-HyA-CS was calculated using Elemental analysis and FTIR.ATR, it was found to be 74%. The adsorption efficiency of the produced adsorbents was compared with pure chitosan to remove of Remazol Brilliant Blue R (RBBR) from an aqueous medium and antimicrobial activity. The removal percentage of RBBR by chitosan, 2-HyA-CS, and 2-HyA-CS/ZnO are 47.12%, 91.9%, and 96.56%, respectively with the following order: 2-HyA-CS/ZnO > 2-HyA-CS > chitosan. Their antimicrobial activities were studied against two Gram negative bacteria ( E. coli and P. aeruginosa), two Gram positive bacteria ( S. aureus and B. cereus) and ( C. albicans) as a yeast strain, the inhibitory zone measurements revealed that the activity of 2-HyA-CS/ZnO is excellent and higher than 2-HyA-CS and pure chitosan. The cytotoxicity of the prepared compound 2-HyA-CS and 2-HyA-CS/ZnO along with pure chitosan was estimated against two human cancer cells MCF-7 cells and HepG-2 cells, the result indicates that 2-HyA-CS/ZnO having higher Inhibitory activity against both MCF-7 and HepG-2 cells with 53.5 ± 2.86 and 27.4 ± 1.23 µg/mL respectively and 2-HyA-CS possessing moderate Inhibitory activity against both MCF-7 and HepG-2 cancer cells with IC50 = 216.5 ± 7.48 and 135.6 ± 6.49 µg/ml respectively.