Chitosan (CS) is a biologically active biopolymer used in different medical applications due to its biodegradability, biocompatibility, and nontoxicity. Nanotechnology is an exciting and quick developing field in medical applications. Nanoparticles have shown great potential in the treatment of cancer and inflammation. In the present work modification of chitosan and its (Ag, Au, or ZnO) nanocomposites by N-aminophthalimide (NAP) occurred through the reaction with epichlorohydrin (ECH) as a crosslinker in the presence or absence of glutaraldehyde (GA) under different reaction conditions using microwave irradiation to give modified chitosan derivatives CS-2, CS-6, and their nanocomposites. Modified chitosan derivatives were characterized using different tools. CS-2 and CS-6 derivatives displayed enhancement of thermal stability and crystallinity compared to chitosan. Additionally, CS-2, CS-6, and their nanocomposites exhibited improvements in antitumor activity against HeLa cancer cells and enzymatic inhibitory against trypsin and α-chymotrypsin enzymes compared to chitosan. However, CS-2 revealed the highest cell growth inhibition% toward HeLa cells (89.02 ± 1.46 %) and the enzymatic inhibitory toward α-chymotrypsin enzyme (17.13 ± 1.59 %). Furthermore, CS-Au-2 showed the highest enzymatic inhibitory against trypsin enzyme (28.14 ± 1.76 %). These results suggested that the new chitosan derivatives CS-2, CS-6, and their nanocomposites could be a platform for medical applications against HeLa cells, trypsin, and α-chymotrypsin enzymes.