The inactivation of antibiotic resistant Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) seeded in greywater by bimetallic bio-nanoparticles was optimized by using response surface methodology (RSM). The bimetallic nanoparticles (Cu/Zn NPs) were synthesized in secondary metabolite of a novel fungal strain identified as Aspergillus iizukae EAN605 grown in pumpkin medium. Cu/Zn NPs were very effective for inhibiting growth of E. coli and S. aureus. The maximum inactivation was optimized with 0.028 mg mL-1 of Cu/Zn NPs, at pH 6 and after 60 min, at which the reduction of E. coli and S. aureus was 5.6 vs. 5.3 and 5.2 vs. 5.4 log reduction for actual and predicted values, respectively. The inactivation mechanism was described based on the analysis of untreated and treated bacterial cells by Field emission scanning electron microscopy (FESEM), Energy Dispersive X-Ray Spectroscopy (EDS), Atomic Force Microscopy (AFM) revealed a damage in the cell wall structure due to the effect of Cu/Zn NPs. Moreover, the Raman Spectroscopy showed that the Cu/Zn NPs led to degradation of carbohydrates and amino structures on the bacteria cell wall. The Fourier transform infrared spectroscopy (FTIR) analysis confirmed that the destruction take place in the C-C bond of the functional groups available in the bacterial cell wall. The techno economic analysis revealed that the biosynthesis Cu/Zn NPs is economically feasible. These findings demonstrated that Cu/Zn NPs can effectively inhibit pathogenic bacteria in the greywater.