Although a large number of different PCR protocols for the detection of fungal DNA from clinical samples have been described, a generally recognised standardisation has not yet been developed. In a first step, we compared six different methods to isolate DNA under in vitro conditions from Aspergillus fumigatus, Candida albicans and Saccharomyces cerevisiae with respect to efficiency and expenditure of time. To this end, methods were tested that are based on both mechanical and enzymatic/thermic lysis. Thereby, enzymatic/thermic lysis were shown to be superior to mechanical lysis, although these methods of DNA isolation were more time consuming. The subsequent comparison of three different PCR protocols showed real-time PCR to be the most sensitive method.