Nanoparticle research is currently an area of intense scientific interest due to a wide variety of potential applications in biomedical, optical, and electronic fields. The biological synthesis of nanoparticles includes the synthesis of nanoparticles using microbes which have the capability to absorb the nanoparticles that exist in various forms. In the biosynthesis of metal nanoparticles by a fungus Botryodiplodia theobromae, the fungus mycelium is exposed to the metal salt solution. The change in color of the medium indicates the synthesis of silver nanoparticles. The synthesized AgNPs were structurally characterized by UV-visible spectroscopy, scanning electron microscope (SEM), and Fourier-transform infrared spectroscopy (FTIR) analysis. The size of the silver nanoparticles was ranging from 62.77 –103 nm in the fungal mat and 66.75–111.23 nm in the cell filtrate respectively with the existence of free OH and NH groups, aromatic CH stretch, C–C stretch, unsaturation in the molecule, and C–Cl stretch. The cytotoxic efficiency of the AgNPs on human MCF7 breast cancer cell lines and A549 human lung cancer cell lines was appraised by cell viability assay. The synthesized AgNPs inhibited the propagation of cells at an IC50 concentration of 100 μg/mL. Thus, this study reveals that the green synthesis is an eco-friendly method for production of AgNPs from endophytic fungi which provided a powerful anti-proliferative action on MCF-7 and A549 cell lines, suggesting them as a novel chemotherapeutic agent against human breast and lung cancers.