The objective of the study was the estimation of quality of organic matter of the humus horizon of soil fertilised every year, for ten years, with spent mushroom (Agaricus L.) substrate, as compared to a soil without such amendment. Fractionation of organic matter was performed, as well as quality analyses of humic acids and fractionation of organic nitrogen complexes. In the humus horizon of the soil fertilised with spent mushroom substrate lower acidity was noted, and higher levels of C, N, P and Ca. In the soil amended with the substrate greater accumulation of soil humus was noted (by nearly 50%), and especially of the fraction of humic acids, which had a positive impact on increasing the value of the ratio of humic to fulvic acids. The fertilisation with the substrate only slightly modified the quality parameters of humic acids. In the soil fertilised with the substrate a higher level of carbon and nitrogen was observed – related with the soluble forms of organic matter, as well as greater accumulation of nitrogen, mainly in a stable non-hydrolysing form.