Colorectal cancer (CRC) is the third major cause of morbidity and mortality worldwide. Hence, many strategies and approaches have been widely developed for cancer treatment. This work prepared and evaluated the antitumor activity of 5-Flurouracil (5-Fu) loaded chromium nanoparticles (5-FuCrNPs). The green biosynthesis approach using Harpullia (H) pendula aqueous extract was conducted for CrNPs preparation that was further loaded with 5-Fu. The prepared NPs were characterized for morphology using scanning and transmission electron microscopes (SEM and TEM). The results revealed the formation of uniform, mono-dispersive, and highly stable CrNPs with a mean size of 23 nm. Encapsulation of 5-Fu over CrNPs with a higher drug loading efficiency was successful with a mean size of 29 nm. In addition, Fourier Transform Infrared (FTIR) and X-ray diffraction pattern (XRD) were also investigated. 5-Fu has been adsorbed on the surface of biosynthesized CrNPs to overcome its clinical resistance and increase its activity against CRC cells. Box-Behnken Design (BBD) and Response Surface Methodology (RSM) were used to characterize and optimize the formulation factors (5-Fu concentration, CrNPs weight, and temperature). Furthermore, the antitumor activity of the prepared 5-FuCrNPs was tested against the CRC cells (CACO-2). The in vitro antitumor study has demonstrated that 5-Fu-loaded CrNPs markedly decreased the IC50 of 5-Fu and exerted more cytotoxicity at nearly all concentrations than 5-Fu alone. In conclusion, 5-FuCrNPs is a promising drug delivery system for the effective treatment of CRC.