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Membrane biofouling limits its lifetime and application. Our objective is to optimize surface modification and subsequent silver nanoparticle impregnation on polyethersulfone (PES) membrane for biofouling prevention. For this, silver nanoparticles were selectively impregnated on the PES membrane, via carbonyl group attachment on membrane surface by acrylic acid grafting. Based on mechanical property and silver loading, three different membranes (AA-Ag-PES-25, AA-Ag-PES-62 and AA-Ag-PES-185 with acrylic acid grafting of 25, 62 and 185 μg cm−2, respectively, and having resultant silver loading of 4.4, 9.06 and 16.94 wt%, respectively) were screened for further testing. Performance of the AA-Ag-PES-62 membrane was best, with least reduction of permeate flux at steady state (12.69%), signifying biofouling prevention, compared to only PES membrane (62.2% permeate flux reduction); the latter due to attached live E. coli cells, in absence of silver nanoparticles. For AA-Ag-PES-25 with a lower silver loading, bacterial cell killing was slower, with resultant permeate flux-drop at steady state (19.7%) being more than the optimum AA-Ag-PES-62 membrane. In contrast, for the higher silver loaded AA-Ag-PES-185, silver nanoparticles formed a surface layer, which resulted in partial pore-blockage with higher permeate flux reduction (27.28%). Regarding long-term performance, for AA-Ag-PES-62, there was neither flux-drop nor bacterial growth till 48 h, whereas, due to low silver content in AA-Ag-PES-25, bacteria re-grew after 24 h, which reflected into permeate flux-drop. AA-Ag-PES-62 membrane provides optimum performance in terms of E. coli killing and anti-biofouling effect, maintaining silver concentration in permeate (29.8 ppb), well within the permissible 100 ppb limit.