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Sustainable fabrication of protective cotton, using bio-extracts, is becoming increasingly attractive. However, many shortcomings—including the introduction of potentially hazardous mordants or modifiers to cotton, annoying colour changes after finishing, and low processing efficiency—require further melioration. Therefore, an efficient ultrasonic-assisted colourless finishing process was developed in this study, to fabricate ultraviolet-proof and antimicrobial cotton. A pair of oppositely charged bio-based substances, i.e., tannin acid (TA) and hydroxypropyltrimethyl ammonium chloride chitosan (HACC) were introduced during the ultrasonic process. The results reveal that cationic HACC significantly promotes the adsorption of TA to cotton. The apparent colour of the cotton remained almost unchanged after finishing. Based on Pesudo first-/second-order kinetic models, chemisorption was verified as the dominant mechanism. Efficiency under ultrasound was enhanced by 5.3% (70 °C) and 27% (90 °C), respectively. A mathematical modelling study established the factors to be in the following order of significance: concentration > pH > temperature. Under optimal conditions, a theoretical maximum UPF of 380.8 was achieved. TA (8 g/L)-treated cotton deactivated up to 98% of Escherichia coli, and also provided excellent UV-shielding performance. In general, the ultrasonic-assisted eco-dyeing and finishing process for cotton was explored in depth from practical and theoretical perspectives, which should push forward the development of the sustainable textile industry. [ABSTRACT FROM AUTHOR]