The present work was designed for the formation of more active antifungal agents via loading copper (Cu) and silver (Ag) nanoparticles (NPs) on a chitosan (CS) natural polymer. Sixty water and sediment samples were collected from Qalubiya, Egypt. One hundred and five fungal isolates were selected according to cultural characteristics, 53 isolates from sediment and 52 from water. Most isolates were identified as Aspergillus sp., Penicillium sp., Candida sp. and Fusarium sp. Nanocomposites (NC) of Cu@CS, Ag@CS, and Cu@Ag@CS were characterized by TEM, SEM, XRD and FTIR analysis for detecting their morphology and size, active surface groups and confirming its conjugation. The average sizes of these nanocomposites were 25, 19 and 33nm, with predominantly spherical shapes in aggregates, respectively. The antifungal study showed that the Cu@Ag@CS nanocomposite (NC) is a more effective and stable antifungal agent. For minimal fungicidal concentration (MFC) Cu@Ag@CS represented the lowest concentrations of 0.125, 0.25, 0.25 and 0.5 mg.ml⁻¹ for Candida sp., Aspergillus sp., Fusarium sp., and Penicillium sp., respectively. In addition, the viable fungal counts (VFCs) of Candida sp. in water treated with Cu@Ag@CS NC was reached to complete inhibition in water after 18 hours, while the cell membrane and cellular contents of treated Candida sp. were destroyed causing cell death. Thus, the loading of Cu and Ag nanoparticles on chitosan proved to form a more active antifungal agent suitable for treating pathogenic fungi in the aquatic environment.