The objective of the work was to evaluate selected properties of spent substrates used for growing button mushrooms (SMSs) and the content and quality of the organic matter in this material in the context of rational use for fertilisation purposes and potential impact on the soil environment. The materials were sampled at production facilities located in the east of Mazovia. The density and amount of spent substrate on shelves where mushrooms were cultivated were determined. The following were analysed in the laboratory: reaction, carbonate content, TC (total carbon) and TOC (total organic carbon) contents, total nitrogen, organic matter fraction composition, and humic acids properties. It was confirmed that this material had a marked potential to enrich soils in organic matter, nitrogen, and carbonates. The analysis revealed that the most important qualitative properties of the organic matter were related to the relatively high share of labile organic compounds (the fraction separated with 0.05 M H2SO4 and the fraction of fulvic acids). The humic acids had similar properties regardless of their origins. The humic acids (HAs) molecules displayed a substantial share of aliphatic structures which are typical of these materials at their initial decomposition stage. It can be assumed that, due to such properties, spent mushroom substrates are materials which can be directly introduced into the soil to improve their quality and prevent degradation.