This study investigated the biosynthesis, statistical optimization, characterization, and biocontrol activity of silver nanoparticles (AgNPs) produced by newly isolated sp. The strain TA-3N was identified based on the ITS gene sequence, together with its phenotypic characteristics (GenBank accession number: OM321439). The color change from light yellow to brown after the incubation period indicates AgNPs biosynthesis. The UV spectrum revealed a single peak with the maximum absorption at 453 nm, indicating that produces AgNPs effectively. A Rotatable Central Composite Design (RCCD) was used to optimize the biosynthesis of AgNPs using the aqueous mycelial-free filtrate of . The optimal conditions for maximum AgNPs biosynthesis (156.02 µg/mL) were predicted theoretically using the desirability function tool and verified experimentally. The highest biosynthetic produced AgNPs by reached 160.3 µg/mL using AgNO concentration of 2 mM/mL, initial pH level of 6, incubation time of 60 h, and biomass weight of 6 g/100 mL water. SEM and TEM imaging revealed uniform spherical shape particles that varied in size between 8.17 and 17.74 nm. The synthesized AgNPs have a Zeta potential value of -9.51 mV. FTIR analysis provided insights into the surface composition of AgNPs, identifying various functional groups such as N-H, -OH, C-H, C=O, and the amide I bond in proteins. Cytotoxicity and genotoxicity assays demonstrated that AgNPs in combination with can mitigate the toxic effects of on barley. This intervention markedly enhanced cell division rates and decreased chromosomal irregularities. The results indicate that AgNPs synthesized by show the potential as an eco-friendly and efficient method for controlling plant diseases. Further studies are necessary to investigate their possible use in the agricultural sector.