The present study aimed to assess the possibility of humic-like substances (HSLs) isolation from spent mushroom substrate (SMS) through alkaline extraction intensified by ultrasound. The Box-Behnken design (BBD) coupled with the response surface methodology (RSM) was applied for the process evaluation. Based on the polynomial model, the values of NaOH concentration, process time, and temperature values were selected to maximize the extraction efficiency. The coefficient of determination for the created model was 97.61 %, and the experimental verification of the response showed that the extraction efficiency under optimal process conditions was equal to 32.66 % ± 1.26 %. Obtained humic-like substances were fractionated into humic-like acids (HLAs) and fulvic-like acids (FLAs) and qualitatively assessed. The ATR-FTIR and CP/MAS 13C NMR spectra of the isolated samples revealed the presence of aliphatic, aromatic, carboxyl and carbonyl structures, which were also evident for the humic and fulvic acids extracted from peat and lignite. Furthermore, HLAs and FLAs were characterized by lower concentrations of heavy metals compared to samples isolated from peat and lignite, which combined with a significant content of micro and macronutrients may indicate the potential to use humic-like and fulvic-like acids extracted from the spent mushroom substrate for agricultural purposes.