Int. J. Environ. Res. Public Health 2010, 7 1. Introduction 3226 An extensive identification and enumeration study of microfungi in a treated municipal water supply system in sub-tropical Australia over an 18 month period showed that microfungi are present throughout the system [1]. Analysis of the treated water leaving the single water treatment plant indicated that the treatment processes are very effective in removing or inactivating microfungal propagules since only occasional microfungal colony-forming units (CFU) were recovered from samples taken at the treatment plant outlet. However, microfungal CFU were routinely recovered from treated water storage reservoirs and distribution mains throughout the distribution system. This finding led to a further investigation to attempt to identify the source(s) of such post-treatment microfungal population. During the collection of monthly water samples from one of the roofed storage reservoirs, designated R3, over an 18 month period it was noticed that numbers of the large Australian green tree frog, Litoria caerulea, were always resident within the reservoir, occupying the internal infrastructure of stairway, supporting columns and roofing beams as diurnal sleeping places. They particularly favoured the sloping C-section side rails of the stairway (Figure 1). Figure 1. Group of Litorea caerulea specimens asleep on a side rail of an internal stairway in a reservoir. Frog excrement was also evident on these structures but it was clear that most faecal pellets are deposited directly into the water or are dislodged into the water by movement of the animals. During the study period a number of unexplained cases of low Escherichia coli counts were reported by a number of water authorities throughout Queensland. The regular observation of L. caerulea within water storage reservoir R3 in the water supply system under study led to the hypothesis that the source of those E. coli counts was the excrement of L. caerulea. Subsequent investigation demonstrated that the frog L. caerulea carries very large E. coli loads [2]. The water authority concerned has since taken steps to exclude L. caerulea from the reservoirs in its water distribution system. Concurrently, it was noticed that old faecal pellets of L. caerulea which remained on the internal structures of reservoir R3 were showing evidence of extensive microfungal growth on their surfaces. This observation suggested three possibilities; (i) that frog excrement contained viable microfungal propagules which germinated post-voidance, (ii) that frog excrement acted as a suitable substrate for the germination and growth of airborne spores which landed on it, or (iii) both. This led to an investigation which aimed to determine whether or not the Australian green tree frog L. caerulea may