Microorganisms are potent contributors to maintaining a safe environment as they are able to degrade organic toxicants. For environmental applications, mostly bacteria are used while fungal strains have received less attention. However, they are able to degrade highly persistent organic contaminants and survive extreme conditions, and may thus be promising organisms. To find new fungal candidates for these applications, twelve soil samples from polycyclic aromatic hydrocarbon (PAH) contaminated sites in Austria were used to isolate fungal strains. A microplate screening method using PAH contaminated soil as inoculant was set up to isolate fungal strains being able to live in presence of toluene, hexadecane, or polychlorinated biphenyl 126. Not many microbial strains are known that degrade these three contaminants, while the PAH contamination acted as selective pressure for the soil microbiota. After obtaining pure cultures, the fungal strains were further screened for their ability to live in the presence of one of the three contaminant substrates. The potential for technical application of the 11 best performing strains, identified using ITS and 18S rDNA, is discussed. The presented microtiter plate screening method is a cost efficient and quick approach to identify fungal strains for pollutant degradation and results in candidates with a high relevance for bioremediation techniques.