Fungi form the largest group of eukaryotic organisms and are widely distributed on Earth. Estimates suggest that at least 1.5 million species exist in nature, yet only 5% have been recovered into pure culture. The fungal diversity of Sub-Antarctic Macquarie Island soil is largely unknown. In this study, a low nutrient fungal culturing approach was developed and used alongside a traditional high nutrient approach to recover Macquarie Island fungi from pristine and a series of Special Antarctic Blend (SAB) diesel fuel spiked soil samples. The low nutrient culturing approach recovered a significantly different (P<0.01) fungal population compared with the high nutrient media approach. In total, 91 yeast and filamentous fungi species were recovered from the soil samples, including 63 yet unidentified species. Macquarie Island has been seriously contaminated by SAB diesel fuel due to the operation of Australia Antarctic research station. Fungi have been known to be able to breakdown hydrocarbons and contribute significantly to bioremediation of soils contaminated with hydrocarbons. Of the 91 recovered fungi species, several were frequently isolated from both medium and high concentrations of diesel fuel contaminated soils and include Antarctomyces psychrotrophicus, Arthroderma sp., Aspergillus sp., Exophiala sp., Geomyces sp., Penicillium sp. and Pseudeurotium bakeri. These dominant species thrived and therefore were tolerant to high concentrations of petroleum hydrocarbons. The ecotoxicological effect of fungal diversity in response to SAB diesel fuel contamination on Macquarie Island soils was investigated on a further three soil plots using the low nutrient culturing approach. A statistically significant difference (P<0.05) in recovered fungal diversity and colony abundance (P<0.001) with increasing concentrations of SAB diesel fuel was observed for examined plots. Fungal colony abundance significantly increased at 50 mg/kg (P<0.001). Thus, SAB diesel fuel was a crucial factor affecting the natural population of fungi present in pristine soil at low concentrations. An EC25 (concentration that results in 25% change from the control response) of 354.81 mg fuel/kg soil for soil bacterial abundance on Macquarie Island was recently determined following SAB contamination, in the future fungal abundance data will also be modeled for EC25 estimations. In this thesis, the diverse fungal species recovered highlights the fact that a routinely adopted at least decade novel cultivation approaches developed for bacteria should be adopted for fungi. A library of fungal isolates from SAB diesel fuel spiked soils is now available that can be characterised further for their potential role in hydrocarbon degradation. The data obtained should be used to towards development of a comprehensive ecotoxicological assessment of Macquarie Island. This site-specific data is important to understand the success of clean-up attempts for contaminated sites and in the future should be incorporated into site-specific management guidelines.