Diversity of microorganisms involved in the biogeochemi-cal N cycle is of fundamental interest in microbial ecology. Denitrifica-tion is a key step in the cycle by which nitrate is reduced to dinitrogen gas via the soluble nitrite and the gaseous compounds nitric oxide and nitrous oxide. The process is carried out by the sequential activity of the nitrate, nitrite, nitric oxide, and nitrous oxide reductase enzyme, respectively. The fluorescence-based quantitative real-time polymerase chain reaction (qPCR) is widely used for quantification of nucleic acids in samples obtained from numerous, diverse sources. Here, we provide a well-proven methodology for isolation of DNA from environmental samples and describe relevant experimental conditions for utilization of qPCR to assay the 16S rRNA and nar/nap, nirK/nirS, c-nor/q-nor, and nos denitrification genes that encode synthesis of denitrifying enzymes. The ISO 11063 standard method and MIQUE guidelines are considered with the aim to increase experimental transparency.