The aim of this study was to determine the properties of organic matter of spent mushroom substrate (SMS) after cultivation of Agaricus bisporus. The research material was obtained after 6 weeks of intensive cultivation. Three crops of the fungus from a modern, high performance mushroom hall were collected. The samples were dried at 40°C and ground in an agate mill. The total carbon (TC) and nitrogen (TN) content were determined on Perkin Elmer’s Series II 2400 autoanalyser.  Fractionation of organic matter was carried out by the Schnitzer method (fractions: after decalcitation, bitumens, fulvic acid, humic acid, residual) and the properties of isolated humic acids were determined (elemental composition, optical properties in the UV-VIS region, Fourier transform infrared spectroscopy FT IR; absorption coefficients, atomic ratios, and internal oxidation states w were calculated). Organic matter of investigated spent mushroom substrates contained large amounts of carbon and nitrogen, and the ratio of these elements was similar to the levels of humus in mineral, biologically active soil. In the analysed SMS most organic carbon was found in the residual fraction (42.5%), less in the humic acids fraction (28.5%), much less in the fulvic acid fraction (14.8%) and the after-decalcitation fraction (11.2%) and the least in bitumens (2.97%). The organic nitrogen content in these organic materials was somewhat different, most of the nitrogen being found in the residual fraction (53.8%), much less in the humic acids fraction (20.4%), after-decalcitation fraction (14.8%) and  fulvic acids fraction (10.6%). Very little content of organic nitrogen was found in bitumens (0.434%). Analysis of the elemental composition of humic acids isolated from organic matter of spent mushroom substrate revealed a relatively high nitrogen content, and the H/C ratio, the UV-VIS optical properties and the IR spectra indicate that these acids are chemically “young”. Bitumens were characterised by high energy value (high hydrogen and carbon content).