Nanotechnology is emerging as one of the most revolutionizing area in research field. Biological methods of reduction of metal ions are often preferred because they are non-toxic, safe, clean, biocompatible and environmentally acceptable. Alternaria brassicae was isolated from rhizospheric region and used for the extracellular synthesis of gold nanoparticles (AuNPs). Further, the study was performed to estimate the antibacterial, antifungal and cytotoxic efficacy of AuNPs. The immunomodulatory efficacy of AuNPs was investigated on various pathogenic microorganisms and human macrophages cell lines (THP1-α). Stable AuNPs were produced when an aqueous solution of chloroauric acid (HAuCl4) was reduced by fungal biomass. Production of nanoparticles was confirmed by the color change from yellow to pinkish violet after approximately 72 hr of reaction. The produced nanoparticles were then characterized by UV-vis spectroscopy, DLS and Transmission electron microscopic analyses. The TEM images of sample revealed that the AuNPs were spherical and hexagonal in shape. Biosynthesized AuNPs were ranged in size from ~15-72 nm. These AuNPs were also assessed for their antimicrobial and cytotoxic efficacy. The results indicated towards efficient antimicrobial activity of AuNPs against Enterobacter aerogene, Bacillus cereus and Trichoderma sp. at higher concentrations. Cytotoxicity analysis of AuNPs on THP1 α cell lines revealed dose dependent behavior. In conclusion, this study proposed the inimitable character of AuNPs prepared from A. brassicae. AuNPs synthesized by this fungus can be either used as cost effective antimicrobial, insecticidal agents or nontoxic immunomodulatory delivery vehicle.