Simple SummaryThe relatively high prevalence of microbial infections and the rising resistance to traditional antibiotics are the causes of the need for revolutionary antibiotics. Nanotechnology, a technique that employs materials featuring nanometer size, has grown in popularity for therapeutic uses and is very intriguing as a means of eradicating or limiting the activity of several pathogens. Silver nanoparticles have been shown to have antimicrobial properties against fungi and bacteria. The unique properties of silver nanoparticles, such as their high surface-area-to-volume ratio and the ability to release silver ions, can cause damage to the microbial cell membrane, interfere with cellular processes, and make them effective against a wide range of microorganisms. Synthesis of nanoparticles via natural products could potentiate their therapeutic activities. Moreover, phosphatase enzyme is also known to possess antimicrobial effects, and there is a fungus (Fusarium oxysporum) reported to have phosphatase enzymes in its extracellular fluid. Therefore, we focused on synthesizing silver nanoparticles by using extracellular proteins released byFusarium oxysporumand thereafter evaluated its biological activities against pathogenic microbes. Our findings illustrated that synthesized nanoparticles showed prominent anti-microbicidal activities against various pathogenic bacterial and fungal species. Thus, these nanoparticles may be used against drug-resistant infections.AbstractNanotechnology based on nanoscale materials is rapidly being used in clinical settings, particularly as a new approach for infectious illnesses. Recently, many physical/chemical approaches utilized to produce nanoparticles are expensive and highly unsafe to biological species and ecosystems. This study demonstrated an environmentally friendly mode of producing nanoparticles (NPs) whereFusarium oxysporumhas been employed for generation of silver nanoparticles (AgNPs), which were further tested for their antimicrobial potentials against a variety of pathogenic microorganisms. The characterization of NPs was completed by UV–Vis spectroscopy, DLS and TEM, where it has been found that the NPs were mostly globular, with the size range of 50 to 100 nm. The myco-synthesized AgNPs showed prominent antibacterial potency observed as zone of inhibition of 2.6 mm, 1.8 mm, 1.5 mm, and 1.8 mm againstVibrio cholerae, Streptococcus pneumoniae, Klebsiella pneumoniaeandBacillus anthracis,respectively, at 100 µM. Similarly, at 200 µM forA. alternata, A. flavus and Trichodermahave shown zone of inhibition as 2.6 mm, 2.4 mm, and 2.1 mm, respectively. Moreover, SEM analysis ofA. alternataconfirmed the hyphal damage where the layers of membranes were torn off, and further EDX data analysis showed the presence of silver NPs, which might be responsible for hyphal damage. The potency of NPs may be related with the capping of fungal proteins that are produced extracellularly. Thus, these AgNPs may be used against pathogenic microbes and play a beneficial role against multi-drug resistance.