During several decades microscopy and culture based methods have been the most important techniques for the detection of fungal infections. Culture, though often slow, sometimes insensitive and sometimes confusing with respect to contamination or colonization, may yield the specific aetiological agent, and may allow susceptibility testing to be performed. However, molecular detection and identification using PCR for the amplification of fungal DNA from tissue is being applied more and more frequently for the early diagnosis and identification of fungal pathogens. Other tools such as fluorescence in situ hybridization (FISH) or DNA microarrays have also been developed and their performance is currently being evaluated. Since standardization and validation for most of these newer techniques are still lacking the combination of various diagnostic tools is still mandatory to allow earlier diagnosis of systemic fungal infections.