Kartchner Caverns, located near Benson, Arizona, USA, is an active carbonate cave that serves as the major attraction for Kartchner Caverns State Park. Low-impact development and maintenance have preserved prediscovery macroscopic cavern features and minimized disturbances to biological communities within the cave.. The goal of this study was to examine fungal diversity in Kartchner Caverns on actively-forming speleothem surfaces. Fifteen formations were sampled from five sites across the cave. Richness was assessed using standard culture-based fungal isolation techniques. A culture-independent analysis using denaturing gradient gel electrophoresis (DGGE) was used to assay evidence of community homogeneity across the cave through the separation of 18S rDNA amplicons from speleothem community DNA. The culturing effort recovered 53 distinct morphological taxonomic units (MTUs), corresponding to 43 genetic taxonomic units (GTUs) that represented 21 genera. From the observed MTU accumulation curve and the projected total MTU richness curve, it is estimated that 51 percent of the actual MTU richness was recovered. The most commonly isolated fungi belonged to the genera Penicillium, Paecilomyces, Phialophora, and Aspergillus. This culturebased analysis did not reveal significant differences in fungal richness or number of fungi recovered across sites. Cluster analysis using DGGE band profiles did not reveal distinctive groupings of speleothems by sample site. However, canonical correspondence analysis (CCA) analysis of culture-independent DGGE profiles showed a significant effect of sampling site and formation type on fungal community structure. Taken together, these results reveal that diverse fungal communities exist on speleothem surfaces in Kartchner Caverns, and that these communities are not uniformly distributed spatially. Analysis of sample saturation indicated that more sampling depth is required to uncover the full scale of mycological richness across spelothem surfaces.