Carbonic anhydrases (CA) are metalloenzymes involved in various physiological and pathological processes, with carbonic anhydrase II (CAII) being the predominant physiological isoform.This study investigates the interaction between CAII and chitosan functionalized iron oxide nanoparticles (C-INPs) as a novel approach to inhibit CAII activity. Iron oxide nanoparticles were functionalized with chitosan of high (HC-INPs), medium (MC-INPs), and low (LC-INPs) molecular weights. The synthesized nanocomposites were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), zeta potential analysis, Fourier-transform infrared spectroscopy (FTIR), and vibrating sample magnetometry (VSM). The average diameters of HC-INPs, MC-INPs, and LC-INPs were approximately 4 nm, 11.5 nm, and 20 nm, respectively, with corresponding Zeta potential values of 39.2 ± 3.9 mV, 36.7 ± 4.1 mV, and 15.5 ± 4.6 mV. Fluorescence quenching assays and enzyme kinetics activity results demonstrated that the interaction of CAII with the nanocomposites induced conformational changes in CAII, inhibiting its enzymatic activity. The inhibitory potential was significantly influenced by the surface properties of the nanoparticles, with HC-INPs showing the most potent effect (Ki = 58.388 ng/ml), followed by MC-INPs Ki = 390.87 ng/ml) and LC-INPs (Ki = 1213.00 ng/ml). These findings suggest that Chitosan-functionalized iron oxide nanoparticles, particularly HC-INPs, represent a promising class of nano-inhibitors for CAII, opening new avenues for treating disorders associated with CAII overexpression.