Worldwide, the environmental nitrate (NO3−) problem is increasingly coming into focus. These increases in NO3− concentration result mainly from agricultural inputs and are further exacerbated by decreasing and finite geogenic NO3− degradation capacity in aquifers. Thus, treatment methods are becoming more and more important. In this study, the effects of enhanced denitrification with addition of organic carbon (C) on thereby autochthonous occurring microbiology and compared at room temperature as well as 10 °C were investigated. Incubation of bacteria and fungi was carried out using natural sediments without degradation capacity and groundwater with high NO3− concentrations. Addition of the four applied substrates (acetate, glucose, ascorbic acid, and ethanol) results in major differences in microbial community. Cooling to 10 °C changes the microbiology again. Relative abundances of bacteria are strongly influenced by temperature, which is probably the explanation for different denitrification rates. Fungi are much more sensitive to the milieu change with organic C. Different fungi taxa preferentially occur at one of the two temperature approaches. Major modifications of the microbial community are mainly observed 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.