Myco-synthesis of silver nanoparticles has garnered considerable interest and has been the subject of research due to their potential in medical diagnostics and treatments. Endophytic fungi that inhabit macroalgae are capable of producing numerous bioactive compounds.  So, in this work, the cell-free aqueous filtrate of the endophytic fungus Trichoderma harzianum OR258299 isolated from the green macroalgae Ulva Lactuca to generate AgNPs. After being visually identified by a color shift, AgNPs were optimized and characterized by Fourier Transform Infrared Spectroscopy (FTIR), Transmission Electron Microscopy (TEM), UV-Vis spectroscopy, and X-ray diffraction (XRD). Antibacterial, antioxidant, and anticancer properties of the AgNPs were examined. The color changed from yellow to brown, suggesting the formation of biogenic AgNPs. FTIR spectra revealed various functional groups, including alcohols, alkanes, unsaturated ketones, aromatic, nitro, and fluorine compounds. Image analysis using TEM identified spherical and ovate-shaped AgNPs with 9.15–32.5 nm diameters. Among the seven separate deviations were observed in the crystalline nature of the AgNPs complex, seven values 1.35938, 1.96175, 2.04762, 2.35792, 2.62036, 2.77221, and 2.95877 were identified.  AgNPs were more effective against Proteus vulgaris ATCC 13315, with an inhibition diameter zone of 19±0.577 mm. According to the DPPH assay, the IC₅₀ values for Ascorbic acid and AgNPs are 10.21 ±0.77µg/ml and 108.07±3.83µg/ml respectively. Cytotoxicity assay demonstrated that AgNPs were highly cytotoxic against breast cancer cells (MCF-7 cell line), with a 50 % inhibitory concentration (IC₅₀) recording 85.97 ± 3.65 µg/ml. The findings demonstrated the encouraging therapeutic application of green synthetic AgNPs.