The progress of nanoparticles production by eco-friendly route, with desirable chemical and physical characteristics, and their application in helpful fields is still under investigation. Therefore, this study aimed at biosynthesis, characterization, and biomedical applications of silver nanoparticles (AgNPs) using yeasts metabolite. The yeast strains, Pichia kudriavzeviiHA-NY2 and Saccharomyces uvarumHA-NY3, were used for extracellular biosynthesis of AgNPsK and AgNPsU, respectively. AgNPs were characterized by UV-visible spectrophotometry, transmission electron microscopy (TEM), Fourier Transformed Infrared (FTIR) spectrum and dynamic light scatter (DLS). TEM image showed well dispersed round and cubic regular particles with size ranges of 12.4 ± 6.02 nm for AgNPsU and 20.655 ± 9.48 nm for AgNPsK. According to DLS analysis, the mean size diameters of AgNPsU and AgNPsK were 20.3-21.5 and 29.6-30.14 nm, respectively. AgNPs showed highly significant inhibitory activity against gram-positive bacteria (Bacillus subtilis ATCC6633 and Staphylococcus aureus ATCC29213), gram-negative bacteria (Pseudomonas aeruginosa ATCC27953), Candida tropicalis ATCC750, and Fusarium oxysporium NRC21. The anti-inflammatory activity of AgNPs revealed that paw edema was inhibited by the oral administration of the two biosynthesized silver-nanoparticles. In addition, they showed carrageenan activity nearest to indomethacin. All fabricated AgNPs showed a significant analgesic effect after one hour of administration, which was comparable to aspirin. Further, both AgNPsK and AgNPsU demonstrated a significant anticancer activity against HCT-116 (Colon cell line) with IC50 values 0.29, 0.24 µg ml, respectively, and PC3 (Prostate cell line) with IC50 values 0.57, 0.50 µg ml, respectively. No ulcerogenic effects of AgNPs were detected on the rats' stomach and it was safe on the gastric profile.