AimsSince mycosynthesis of metal nanoparticles (NPs) is advertised as a promising and ecofriendly approach. Thus, this study aims to investigate the capability of Aspergillusbrasiliensis ATCC 16404 for mycosynthesis of silver NPs (AgNPs). Methods and ResultsOne-factor-at-a-time-technique was used to study the effect of different physicochemical parameters: the reaction time, pH, temperature, different stirring rates, illumination, and finally, the different concentrations of silver nitrate and fungal biomass on the mycosynthesis of AgNPs. The visual observation showed the characteristic brown colour formation due to the bioreduction of Ag+ ions to Ag-0 by the mycelial cell-free filtrate (MCFF). The UV/visible spectrophotometric technique displayed a characteristic sharp peak at ?(440) confirming the mycosynthesis of AgNPs. The zeta potential value -167mV assured the long-term stability of AgNPs and the dynamic light scattering analysis revealed good dispersion and average particle size 77nm. The energy dispersive X-ray spectroscopy displayed a maximum elemental distribution of silver elements. The X-ray diffraction spectroscopy demonstrated the crystallinity of the mycosynthesized AgNPs. The field emission scanning electron microscope and high-resolution transmission electron microscope revealed monodispersed spherical shaped AgNPs with average particle size of 6-21nm. The FTIR analysis showed the major peaks of proteins providing the possible role of MCFF in the synthesis and stabilization of the AgNPs. The mycosynthesized AgNPs expressed good biocidal activity against different pathogenic micro-organisms causing some water-related diseases and health problems to local residents. ConclusionsThis study proved that A. brasiliensis ATCC 16404 MCFF has good potential for mycosynthesis of AgNPs, which exhibited good antimicrobial effect on different pathogenic micro-organisms; thus, it can be applied for water disinfection. Significance and Impact of the StudyThis research provides a helpful insight into the development of a new mycosynthesized antimicrobial agent.