PCR is a very appealing technology for the detection of human pathogens, but the detection of fungal pathogens is particularly challenging. Fungi have cell walls that impede the efficient lysis of organisms and liberation of DNA, which can lead to false-negative PCR results. Conversely, some human pathogens are also ubiquitous environmental saprophytes that can contaminate PCR reagents and cause false-positive results. We examine the quality of PCR-based studies for fungal diagnostics using 42 variables within the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines. This review focuses on taxon-directed PCR assays for the diagnosis of invasive aspergillosis, candidiasis and Pneumocystis pneumonia. Finally, we evaluate broad-range fungal PCR assays capable of detecting a wide spectrum of human pathogens.