Fungi constitute an important group, not only in the biological diversity of soils, but also as a public health concern because of their role in causing disease in humans. Analysis of natural or invasive disease causing populations requires high rates of sampling, due to the high number of fungal species involved in natural systems, or in a hospital setting. The aim of this study was to develop a rapid, high-throughput application for processing and analysing large numbers of fungal samples. We utilized specific ribosomal DNA internal transcribed spacer (ITS) sequences to screen environmental samples by PCR. Current methods used for the identification of fungal species either involve time consuming and labour intensive DNA extractions prior to analysis, or culture-dependent methods of identification and diagnosis of fungal infections. Molecular methods allow for the development of better ecological fungal databases, through genotyping and expanding the current knowledge of disease-causing fungal species. We have developed a ‘quick turnaround’, streamlined procedure of DNA extraction followed by subsequent PCR amplification and analysis. This system is highly adaptable to an automated system for rapid DNA extraction and amplification.