The mesophilic and psychrotolerant microbiota of the air, soil, water, and bottom sediments of the Kinderlinskaya cave (South Urals, Russia) and the factors affecting the structure of microbial communities were investigated. The pattern of microbial distribution in soils was shown to depend on both the configuration of the cave and the level of recreational load. The lowest numbers of bacteria and micromycetes were found in the poorly visited, difficult-to-access sites. Coliform bacteria were revealed in all soil and sediment samples and in some water samples. Micromycetes belonged to 19 genera, with Geomyces pannorum as the dominant species. Air movement was shown to be the main factor affecting the density of the aerial microbiota.