The present research aims to produce gold nanoparticles (AuNPs) from the aqueous extract of locally isolated mushroom Coprinus comatus from Hit city, Iraq. Its properties were studied using the optical vision, UV-Vis, EDX, XRD, FTIR, AFM, and Zetasizer analyses. The exposure of the colloidal solution of AuNPs to UV radiation was investigated for 1, 2, and 3 h. The results showed the color change of the interaction mixture from light yellow to purple after 25 min. The lambda max of the absorbance reached 530 nm using UV-Visible spectrum as evident in the formation of AuNPs. FTIR spectra revealed the presence of functional groups related to peptides, proteins, flavonoids, monosaccharides, and phenolic compounds, which reduced gold ions. The EDX technique showed that the formed nanoparticles were AuNPs. XRD results showed that AuNPs have a face-centered cubic (fcc) crystal. The UV irradiation at different times led to an increase in the intensity of absorbance and sizes of AuNPs from 17.39 nm before the irradiation and switched to 58.16, 59.13, and 47.35 nm after 1, 2, and 3 h, respectively, but their sizes remained within the nanoscale range (less than 100 nm). In conclusion, the best result was observed after about an hour on the effects of UV irradiation on sizes of AuNPs, which reached smaller nanoparticles compared with times 2 and 3 h.