The article is devoted to the IBK Mushroom Culture Collection as an essential basis of preservation of genofund of macrofungi in pure culture and fundamental research on biology and biotechnology of edible and medicinal mushrooms in Ukraine. Isolation methods of macromycetes pure cultures are described and nutrient agar media to preserve the vegetative mycelium in a viable state are listed. The verification methods using the DNA nucleotide sequences are described, cultural morphological features of the strains on agar nutrient media and microstructures of vegetative mycelium by optical and scanning electron microscope are characterized. Production of biologically active substances - melanins, polysaccharides, lipids, cytokinins, antimicrobial activity, and the ability of heavy metal sorption by numerous macromycetes was studied. A list of some important for biotechnology edible and medicinal mushroom species maintained in the IBK Collection is provided. A special attention paid in the IBK Collection to cultures of rare fungi species, particularly of those listed in the Red Data Book of Ukraine. Criteria of their identification and verification are briefly discussed. Microscopic features of vegetative mycelia and morphological characters of the colonies of 10 rare for Ukraine fungi species are given. The main parameters of nutrient media, plant biostimulants and cultivation conditions for certain types of macromycetes are characterized. The strains of the IBK Collection, potential producers of biologically active substances, were selected and their cultivation conditions, composition of nutrient media and methods for stimulating the synthesis of biologically active compounds have been established. High-yielding edible and medicinal mushrooms strains for fruit bodies’ production on the elective plant substrates were demonstrated.