A spent mushroom substrate (SMS) was mixed with wheat straw (WS) in three proportions, C1 (2:1), C2 (4:1), and C3 (6:1), and composted for 90 days in static piles with periodic turning to ensure adequate aeration. Samples from each pile were collected periodically (after 0, 30, 60, and 90 days), and the humic acid-like fractions (HAs) were isolated to determine their elemental composition (C, H, N, S, and O), acidic functional group (carboxylic and phenolic) content, and structural and functional characteristics using spectroscopic methods including ultraviolet-visible, Fourier transform infrared (FTIR), and fluorescence. The results of elemental and functional group analyses show that, with increasing time of composting, the N, O, and acidic functional group contents of HAs increase, whereas their C and H contents and C/N ratio decrease. The analysis of FTIR and fluorescence spectra shows that, with increasing composting time, the presence of aliphatic and polysaccharide-like structures in HAs decreases, whereas oxygenation, polycondensation, and polymerization increase. These results suggest that the chemical and structural characteristics of the HA fractions in the final composts resemble those typical of native soil HAs, which indicate that an adequate degree of maturity and stability is achieved after the end of composting. The results of the present study confirm that composting is an appropriate treatment to transform fresh organic matter (OM) in SMS into humified forms, thus enhancing their quality, agronomic efficiency, and environmental safety as a soil OM resource for application as soil amendment.