Curvularia spicifera (SUMCC 22003) is an endophytic fungus isolated from leaves of the medicinal plant Calotropis procera that were collected from Wadi Bir-EL-Ain, Sohag, Egypt. The fungus was identified based on morphology and phylogenetic analyses. The ability of C. spicifera to biosynthesize silver nanoparticles (AgNPs) was studied. The biosynthesized AgNPs were characterized using UV-vis spectroscopy, XRD measurement, DLS, Zeta potential analysis, FTIR and HR-TEM analysis. The formed AgNPs were stable, well-dispersed and spherical crystalline with an average diameter of 38.41 nm and a Zeta potential of -6.35 mV. The FTIR analysis confirmed that AgNPs are capped with protein. The biosynthesis optimization study indicated that 1 mM AgNO3, 5 g of biomass weight, pH 10.5 and a reaction temperature of 60°C were the optimal conditions for AgNPs biosynthesis. AgNPs exerted significant antimicrobial activity at different concentrations against the tested species of Gram-negative bacteria, Gram-positive bacteria and yeast species, demonstrating their potential as broad-spectrum antimicrobial agents. Escherichia coli showed the highest susceptibility to AgNPs (50 µg) with an inhibition zone diameter of 23.7±0.3 mm and MIC 4.2±0.1 µg. AgNPs (50 µg) exhibited an inhibition zone of 16.7±0.1 mm and MIC of 5.7±0.3 for Candida albicans.