Cupryl species (Cu(III)) are promising oxidants for degrading recalcitrant organic contaminants and harmful microorganisms in water. In this study, defect-rich cuprous oxide (D-Cu₂O) nanospheres (NSs) are introduced as a Fenton-like catalyst to generate Cu(III) for the inactivation of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). D-Cu₂O, in the presence of H₂O₂, achieved inactivation efficiencies 3.2, 3.0, and 2.4 times higher than those of control Cu₂O for ARB, extracellular ARGs (e-ARGs), and intracellular ARGs (i-ARGs), respectively. Experimental evidence from oxidant scavenging tests, Cu(III)-periodate complexation assays, electron paramagnetic resonance (EPR), and in situ Raman spectroscopy confirmed that D-Cu₂O significantly enhanced Cu(III) generation when reacting with H₂O₂ compared to control Cu₂O. Density functional theory (DFT) calculations further revealed that unsaturated copper atoms in D-Cu₂O enhance H₂O₂ adsorption by improving the structural accessibility of adjacent oxygen atoms. This facilitates electron transfer processes and promotes subsequent Cu(III) generation. The D-Cu₂O/H₂O₂ system demonstrated excellent reusability, maintaining a 4-log reduction of ARB over five cycles, and proved effective across various water matrices and microbial species. These findings highlight the potential of the D-Cu₂O/H₂O₂ system, driven by defect engineering, as a robust platform for enhancing water safety and advancing sustainable disinfection technologies.