The polymerase chain reaction is a powerful molecular tool for the detection and analysis of very small amounts of DNA. Today, hybridization probes are often used in real-time PCR for more sensitive and specific detection of pathogens and for determination of gene regulation or mutation analysis instead of intercalating dyes like SYBR Green. Here, we describe how to generate suitable primers and hybridization probes for the specific detection of fungal DNA. Furthermore, we show the advantages of hybridization probes using the LightCycler-PCR for the detection of different Candida spp. and Aspergillus spp. in patient blood samples. The methods used to develop such PCR assays will also be presented in the following protocol.