Herein, a series of vanillin-crosslinked chitosan (Vn-CS) nanocomposites (NCs) containing various contents of ZnO nanoparticles (NPs) was prepared and characterized via FTIR spectroscopy, XRD, TGA, SEM and TEM. Changing the weight% of ZnO NPs in the prepared NCs resulted in an improvement in their antibacterial activity against Gram-negative and Gram-positive bacteria strains compared with the unmodified CS, and the encapsulation efficiency of 5-fluorouracil (5-FU) was found to be in the range of 61.4–69.2%. Subsequently, the release of 5-FU was monitored utilizing the mesoporous ZrO₂–Co₃O₄ NPs modified carbon paste sensor via the square-wave adsorptive anodic stripping voltammetry (SW-AdASV) technique. Also, the release mechanism of 5-FU from each NC was studied by applying the zero-order, first-order, Hixson–Crowell and Higuchi models to the experimental results. The cytotoxicity of prepared NCs and 5-FU-encapsulated NCs was evaluated against the HePG-2, MCF-7 and HCT-116 cancer cell lines, in addition to the WI-38 and WISH normal cell lines using the MTT assay. Notably, 5-FU/CV₁₀ NC exhibited the highest antitumor activity towards all tested cancer cell lines and a moderate activity against WI-38 and WISH normal cell lines with IC₅₀ values of 28.02 ± 2.5 and 31.65 ± 2.7 μg mL⁻¹, respectively. The obtained nanocomposites exhibited suitable selectivity with minimum toxicity against normal cells.