GREEN synthetic strategies have been receiving a great interest for metal nanoparticles synthesis. In the current study, extracellular synthesis of silver nanoparticles (AgNPs) using the cell filtrate of the endophytic fungus, Alternaria tenuissima AUMC 13621 isolated from healthy leaves of Ruta graveolens plant, was achieved. The biosynthesized AgNPs were characterized by UV–visible spectroscopy, transmission electron microscopy (TEM), dynamic light scattering analysis (DLS), and fourier transform infra-red (FT-IR), The UV–visible spectrum shows a maximum absorption peak at 416nm. TEM photography showed the spherical shape of AgNPs with an average size 9.8nm. FT-IR indicates bounding of silver nanoparticles with the supernatant molecules of A. tenuissima and provides evidence for the presence of proteins and biomolecules responsible for reduction of Ag+ to Ag0, capping and stabilizing the synthesized AgNPs. Parametric optimization for the biosynthesis process showed maximum absorbance of 400–408nm at pH-12, 70oC using 1mM AgNO3 concentration. By studying the effect of gamma irradiation on AgNPs biosynthesis, the results showed that, the obtained peak became more sharper and narrower moreover, the absorbance intensity increased by increasing irradiation dose from 0.5 to 3.0kGy. The synthesized Ag NPs have a potent antimicrobial activity to some tested pathogenic bacterial strains and Candida albicans ATCC1023. Additionally, they had excellent free radical scavenging activity. The AgNPs antioxidant activity at 100μg/ml was of 65.99% inhibition that was close to the result obtained for ascorbic acid (67.39%).