ZnO-NPs have been recently recognized as a potent antibacterial agent used in medical textiles due to its activity against a broad spectrum of pathogenic bacteria. Herein, in situ deposition of ZnO-NPs onto cotton fabrics was formulated in a biological manner by the use of secreted fungal enzymes and proteins. Fungus Pacelomyces sp. AF-1 was harnessed in the preparation of ZnO-NPs via the use of its biomass filtrate as a medium for the deposition of ZnO-NPs onto cotton fabrics in the presence of zinc nitrate and NaOH as an alkali. Thorough chemical and physical investigations were conducted, including FTIR, XRD, TEM, UV-Vis spectra, Particle size, zeta potential and antibacterial activity. Results clearly revealed the successful deposition of ZnO-NPs onto cotton fabrics with size ranging from 5 to 20 nm with spherical well stabilized narrow size particles. By increasing the concentration of Zn(NO3)2 up to 1.0 mM, the size of the formed ZnO-NPs increased indicating the low stability of the formed ZnO-NPs. In addition, results also affirmed the antibacterial activity of cotton fabrics deposited ZnO-NPs against E. coli and S. aureus with high durability that kept their antibacterial activity after 15 washing cycles with bacterial reduction of about more than 50% which is considered as a highly durable antibacterial cotton fabric.