Nanoparticles (NPs) are encapsulating agents that exist in the nanometer range. They can be classified into different classes based on their properties, shapes, or sizes. Metal NPs, fullerenes, polymeric NPs, ceramic NPs, and luminescent nanoporous hybrid materials are only a few examples. This study explored the anticancer potential of quercetin and 5-fluorouracil-encapsulated chitosan nanoparticles (CS-5-FU-QCT NPs). The nanoparticles were prepared by ionic gelation, and their efficacy and mechanism of action were examined. CS-5-FU-QCT NPs were characterized using dynamic light scattering (DLS), atomic force microscopy (AFM), UV-visible spectroscopy, and Fourier transform infrared spectroscopy (FTIR); cytotoxicity was analyzed using an MTT assay. Cells were treated with CS-5-FU-QCT NPs and incubated for 12, 24, and 36 h, and apoptosis analysis (using Annexin V/FITC), cell-cycle analysis, Western blotting, and confocal microscopic analysis were performed. Biophysical analysis revealed that the CS-5-FU-QCT NPs fall in the range of 300-400 nm with a near-spherical shape. The i drug release profile indicates sustained release of drugs over a period of about 36 h. The cytotoxicity of CS-5-FU-QCT NPs was more prominent in HCT116 cells than in other cancer cells. This particular nanoformulation caused G0/G1 phase cell-cycle arrest in HCT116 cells and induced intracellular ROS generation, thereby causing apoptosis. It also downregulated Bcl2, cyclin D1, and Cdk4 and upregulated BAX, p53, and p21, causing cell-cycle arrest and apoptosis. In summary, CS-5-FU-QCT NPs hindered proliferation of HCT116 cells via ROS generation and altered the expression of key proteins in the p53/p21 axis and apoptotic machinery in a time-dependent manner.