Gold nanoparticles (GNP) were bio-fabricated utilizing the methanolic extract of the endophytic isolate Aspergillus terreus. The biosynthesised gold nanoparticles (GNP023) were characterised using UV-visible spectroscopy (UV-Vis); transmission electron microscopy (TEM), Fourier-transform nfrared spectroscopy (FTIR) and X-ray diffraction (XRD) studies. The bio-fabricated GNP023 displayed a sharp SPR peak at 536 nm, were spherically shaped, and had an average size between 10–16 nm. The EDX profile confirmed the presence of gold (Au), and XRD analysis confirmed the crystalline nature of GNP023. The antimicrobial activity of GNP023 was investigated against several food-borne and phytopathogens, using in vitro antibacterial and antifungal assays. The maximum zone of inhibition was observed for S. aureus and V. cholera at 400 μg /mL, whereas inhibition in radial mycelial growth was observed against Fusarium oxysporum and Rhizoctonia solani at 52.5% and 65.46%, respectively, when challenged with GNP023 (200 μg/mL). Moreover, the gold nanoparticles displayed significant antioxidant activity against the ABTS radical, with an IC₅₀ of 38.61 µg/mL, and were non-toxic when tested against human kidney embryonic 293 (HEK293) cells. Thus, the current work supports the application of myco-synthesised gold nanoparticles as a versatile antimicrobial candidate against food-borne pathogens.