Green-synthesized gold nanoparticles demonstrate several therapeutic benefits due to their safety, non-toxicity, accessibility, and ecological acceptance. In our study, gold nanoparticles (AuNPs) were created using an extracellular extract from the fungus Schizophyllum commune (S. commune). The reaction color was observed to be a reddish pink after a 24 h reaction, demonstrating the synthesis of the nanoparticles. The myco-produced nanoparticles were investigated using transmission electron microscopy (TEM), dynamic light scattering (DLS), and UV–visible spectroscopy. The TEM pictures depicted sphere-like shapes with sizes ranging from 60 and 120 nm, with an average diameter of 90 nm, which is in agreement with the DLS results. Furthermore, the efficiency of the AuNPs’ antifungal and cytotoxic properties, as well as their production of intracellular ROS, was evaluated. Our findings showed that the AuNPs have strong antifungal effects against Trichoderma sp. and Aspergillus flavus at increasing doses. Additionally, the AuNPs established a dose-dependent activity against human alveolar basal epithelial cells with adenocarcinoma (A549), demonstrating the potency of synthesized AuNPs as a cytotoxic agent. After 4 h of incubation with AuNPs, a significant increase in intracellular ROS was observed in cancer cells. Therefore, these metallic AuNPs produced by fungus (S. commune) can be used as an effective antifungal, anticancer, and non-toxic immunomodulatory delivery agent.