The diversity of groundwater mycobiota remains relatively unknown and unchartered. As a first description of the fungal diversity in Australian aquifers we explored the distribution, abundance and diversity of fungal assemblages in samples from an alluvial (Bylong) and two sandy (Tomago; Botany Sands) unconfined aquifers in coastal catchments of NSW, Australia. A total of 89 strains were isolated with 54 Eumycotan taxa identified. The Botany Sands aquifer, with a history of industrial chlorinated hydrocarbon contamination along a hydraulic gradient, yielded the highest richness with 23 identifiable taxa, followed by Tomago with 17 and Bylong with 14. Despite differences in geology and land use, the aquifers shared similarities in their fungal assemblages with Penicillium, Rhodotorula, Paecilomyces and Cladosporium spp. common to all sites. Fungal assemblages were notably similar between the Bylong and Tomago samples and differed from the Botany samples in composition and heterogeneity. Taxa appeared to cluster into two main groups according to their association with environmental conditions; with the taxa common to the Botany aquifer correlating strongly with higher dissolved oxygen concentrations. The diversity and spatial heterogeneity of groundwater mycobiota appeared to be influenced by both anthropogenic contaminants and associated environmental variables. Based on the criteria of ubiquity, abundance and potential sensitivity to contamination, Penicillium and Rhodotorula spp. might be fitting taxa for monitoring groundwater quality.