Background: Colorectal cancer (CRC) is among the most diagnosed malignancies, accounting for 7.6% of new cancer cases (2024). Standard treatments such as surgery, chemotherapy, and radiation are limited due to the development of drug resistance, characterised by enhanced drug efflux and DNA repair activity. Chemotherapeutic drugs such as 5-fluorouracil, oxaliplatin, and irinotecan induce cytotoxicity by disrupting DNA synthesis/causing DNA damage. However, cancer cells can counteract these effects by activating multiple DNA repair pathways (NER, MMR, BER, HR, and NHEJ), as well as overexpression of drug transporters (MRP1). This study aims to develop peptide-functionalised metallic nanoparticles that target the DNA repair pathways (NER, MMR, HR, NHEJ) and the MRP1 drug transporter to enhance therapeutic sensitivity. Peptide-functionalised gold nanoparticles targeting NHEJ and NER were successfully prepared and characterised by a sharp absorbance peak at 530 nm. Experimental optimisation was conducted using MRC5VA and HCT-116 cells, with radiation treatment (0–8 Gy). Clonogenic assays confirmed that irradiated cells formed fewer colonies than untreated cells, indicating radiation-induced DNA damage halted cell division. In HCT-116 cells, nanoparticle treatment alone was non-toxic across concentrations (1×10⁶–5×10⁷ nanoparticles/cell), yet combining nanoparticles with radiation significantly reduced colony formation in a dose-dependent manner, demonstrating the effective inhibition of DNA repair pathways. γH2AX immunofluorescence staining depicted higher DNA damage in HCT-116 cells compared to MRC5VA due to genomic instability, while flow cytometry confirmed radiation-induced cell-cycle arrest.