Harmanecká Cave is located in the Harmanec Valley to the northwest of Banská Bystrica city, in the southern part of the Great Fatra Mountains, Slovakia. This cave is the most important underground locality of bat occurrence in Slovakia (population of 1000 to 1500 individuals). The study aimed at mycological evaluation of the air, the water, and the rock surface of Harmanecká Cave in Slovakia. The samples were taken on 24 July 2014. To examine the air, the Air Ideal 3P sampler was used. Microbiological evaluation of the rock surface was performed using swab sampling and the water by using the serial dilution technique. The authors observed a relationship between air temperature and the concentration of fungi. The concentration of airborne fungi increased with the increase in the air temperature and decreased with distance from the entrance to the cave. The density of airborne fungi isolated from the outdoor air samples was 810.5 colony-forming units (CFU) per 1 m3 of air and from 27.4 to 128.5 CFU for the indoor air samples. From the rock surface inside the cave, 45.0 to 106.6 CFU per 1 cm2 were isolated, whereas from the water, 29.9 CFU per 1 ml were isolated. Seven species of filamentous fungi were isolated from the external air samples and 12 species of filamentous fungi and 3 species of yeast-like fungi from the internal air samples. From the surface of the rocks inside the cave, 5 species of filamentous fungi and 1 species of yeast-like fungi were cultured, whereas from the water samples, 6 species of filamentous fungi were cultured. Cladosporium spp. were the fungi most frequently isolated from the external air; from the internal air, Penicillium urticae was most frequently isolated; from the rock surface, it was Gliocladium roseum; and from the water, it was P. chrysogenum. The species found in the cave can be pathogenic for humans and animals, especially for immunocompromised persons, and they can also cause biodegradation of the rocks. However, the concentration of airborne fungi inside the cave did not exceed official limits and norms stated as dangerous for the health of tourists.