This study aimed to synthesize gold nanoparticles (AuNPs) by hot water extract in room conditions using edible Morchella esculenta (ME) and investigate the bioactive properties of the synthesized Morchella esculenta-based gold nanoparticles (ME-AuNPs). The characterization of the biologically synthesized ME-AuNPs was made using the ultraviolet–visible spectrophotometry, X-ray crystallography, scanning electron microscopy, Fourier transforms infrared spectroscopy, and energy dispersive X-ray spectrum methods. The ME-AuNPs, with a particle size of 16.51 nm, were found to have strong bioactive properties. The antioxidant activity of the ME-AuNPs attempted by metal chelating activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and the β-carotene linoleate model system. The activities at 10 mg/mL were 82, 85, and 77% for the chelation of ferrous ions, DPPH scavenging, and β-carotene linoleate tests, respectively. The ME-AuNPs also showed strong antimicrobial activity against various pathogen microorganisms and strong cytotoxic activity in the A549 and HepG2 cell lines. This study demonstrated the possibility of using a cheap and nontoxic fungal extract as a reducing and stabilizing agent for the synthesis of size-controlled, large-scale, and biocompatible AuNPs that could be used in future diagnostic and therapeutic applications
