A broad spectrum, total of 195 microfungal taxa, were isolated from various cave substrates (cave air, cave sediments, bat droppings and/or guano, earthworm casts, isopods and diplopods faeces, mammalian dung, cadavers, vermiculations, insect bodies, plant material, etc.) from the cave system of the Domica Cave (Slovak Karst National Park, Slovakia) using dilution, direct and gravity settling culture plate methods and several isolation media. Penicillium glandicola, Trichoderma polysporum, Oidiodendron cerealis, Mucor spp., Talaromyces flavus and species of the genus Doratomyces were isolated frequently during our study. Estimated microfungal species diversity was compared with literature records from the same substrates published in the past.