This protocol was used for the molecular profiling of the soil fungal community of two winter wheat fields as a part of a long-term field trial in Germany with following Illumina Amplicon Sequencing. Besides different pre-crops, the effects of soil management and fertilization on the fungal communities were investigated. Due to different aspects of our protocol, we wanted to ensure reproducing a realistic ratio of the different fungal communities in the soils. The most important steps to conform this demand being the frequently measurements of DNA concentrations at different steps of the protocol, the high numbers of PCRs of each soil sample and the use of the qPCR setting a cut-off for the PCR cycles to avoid an over-amplification of certain PCR-fragments and a loss of detectable biodiversity. The use of the two DNA isolation kits based on the cooperation with our project partner and the exchange of DNAs.