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Nanofinishing is the process by which ultrafine dispersion of nanomaterials is applied to a textile for the development of functionalities. The utilization of nanometal oxides as antimicrobial agents have shown a substantial antimicrobial property in cotton. In the present study, previously synthesized powder containing ZnO nanoflowers (ZnO NFs) was characterized for morphology, surface composition, roughness, and charge using Transmission electron microscopy (TEM), Scanning transmission electron microscopy (STEM), Atomic force microscopy(AFM) and Zeta potential. Optical properties of crystalline ZnO were determined by Photoluminescence (PL), Diffused reflectance Spectroscopy (DRS), and bandgap energy determination. Highly crystalline, ZnO NFs bearing crystal defects and high surface charge were loaded onto the pristine cotton by a dip coating method using Triton X-100 as dispersant and iSys MTX fabric binder. The pristine cotton fabric of 125 g/m2 was nano finished by loading 20,42 and 58 µg/cm2 (1–3 dip cycles) ZnO NFs respectively. The loading of ZnO NFs onto the surface of cotton fabric was confirmed by SEM and used for antibacterial activity against E. coli as a photocatalytic reactor. The prepared samples were irradiated with a UV lamp of λmax = 254 nm (15 min, 30 min, 45 min) and D65 artificial sunlight (60 min, 120 min, 180 min) to investigate their photocatalytic activity against pathogenic E. coli using modified Breed Smear’s method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ZnO NFs@ cotton were determined as 19.53 µg/ml and 39.06 µg/ml respectively after exposure to UV light. After exposure to sunlight MIC and MBC observed were higher i.e. 156.25 µg/ml and 312.5 µg/ml respectively showing lesser activity in sunlight as compared to ionizing UV radiations. To verify the photocatalytic activity, hydroxyl radicals generated by ZnO NFs@ cotton were also determinedtime-resolved PL on exposure to a UV lamp and D65 artificial sunlight. This nano-finished cotton is a promising candidate to be used as a medical textile with high antibacterial activity even after 20 washing cycles with only a 5% decrease in efficiency.