Abstract Significant effects of two fractions of alkali-extractable soil organic matter (AEOM) extracted from three different soils (agricultural soil, soil from deciduous forest, soil from spruce monoculture) on mycelial growth of 17 isolates of ectomycorrhizal fungi were observed. Growth of Lactarius deterrimus, Meliniomyces bicolor and one of the isolates of Leccinum aurantiacum was significantly stimulated by acid-insoluble fraction extracted from all three soils. The stimulatory effects were frequent but inhibition of mycelial growth of some isolates was also observed. The fungal response to the presence of the organic extract in the nutrient medium was isolate-specific rather than species-specific. Organic matter extracted from different source soils affected differently the mycelial growth, the largest number of stimulatory effects being observed in an experiment where the extract was richest in trace elements Zn and Cu. At the same time, the observed stimulatory effects were not attributable to increased concentrations of trace elements in the nutrient medium. The results indicate that soil may be used as a source of extractable organic fractions which, when used as a cultivation medium additive, may significantly improve the growth of responsive fungal isolates. Under natural conditions, AEOM (traditionally designated humic substances) represent a potential factor affecting the composition of cenosis of ectomycorrhizal fungi in soil.