<p>Traditional copper oxide nanoparticles (CuONPs) for antimicrobial textiles face significant challenges. First, the synthesis is reagent-intensive, requiring hazardous substances such as sodium hydroxide. Second, the resulting CuONPs usually exhibit poor aqueous dispersibility and low affinity for fabrics. Herein, an amino-terminal hyperbranched poly(amide–amine) and Cu²⁺ complex (HBPAA-Cu²⁺) was developed. This complex can be coated to cotton fabric via a conventional dipping-rolling-drying process and converted in situ into HBPAA-capped CuONPs (HBPAA-CuO) through thermal oxidation. This approach avoided the need for additional alkaline precipitants and surfactants during the formation of CuO and was expected to reduce the amount of CuONP-containing wastewater generated compared to conventional wet-chemical synthesis routes. Owing to the combined effect between the antimicrobial cationic HBPAA and the CuONPs, no colonies of E. coli and S. aureus were detected for the treated fabric even after 50 laundering cycles (Cu content &gt; 100&#xa0;mg/kg). Furthermore, cell viability remained above 80% with Cu content below 300&#xa0;mg/kg, indicating low cytotoxicity. The treatment also preserved the fabric’s original wearability, including tensile strength, air permeability, and flexural stiffness. This strategy offers a facile and sustainable pathway for the large-scale production of washable, low-cytotoxicity, and antimicrobial textiles.</p>