Worldwide, the nitrate (NO3-) problem and rising NO3- pollution are increasingly coming into focus. These increases in NO3- concentration result, among others, from decreasing and finite geogenic NO3- degradation capacity in aquifers. Thus, treatment methods are becoming more and more important. In this study the microbiology was investigated, which was achieved by an enhanced denitrification with addition of organic carbon (C). Incubation of the investigated bacteria and fungi was carried out in batch tests, with natural sediments without degradation capacity and groundwater with high NO3- concentrations. For the first time, the occurrence of the microbial community influenced by enhanced denitrification is compared at room temperature and 10 °C (typical groundwater temperature). Addition of the four applied substrates (acetate, glucose, ascorbic acid, and ethanol) results in a large change in the microbial community compared to the zero sample. In addition, cooling to 10 °C changes the microbial community again with the same experimental procedure and substrate application. The general appearance of the bacteria is independent of temperature. However, the relative abundances are strongly influenced. Thus, denitrification rates are also decisively influenced by temperature. Fungi are much more sensitive to the milieu change with organic C. Similarly, fungi suffer from cooling to 10 °C. Therefore, fungi preferentially occur at one of the two temperature approaches. The strong change in the microbial community, is mainly observed in the samples whose denitrification rates strongly depend on the temperature effect. Therefore, we assume a temperature optimum of enhanced denitrification specific to each substrate, which is influenced by the microbiology.