The aim of this work was to study freshwater Nitrososphaeria and their impact on the nitrogen cycling in lakes. We followed their absolute abundance in oligotrophic Lake Constance (Germany) with quantitative polymerase chain reaction (qPCR) over a time frame of two consecutive years from November 2017 until November 2019. During this time we took water samples monthly (2017/2018) or as required (2019) at a depth of 85 m. We combined this data with measurements of nitrate and ammonia concentrations in the whole water column. In addition, we performed stable isotope experiments with addition of 15N-ammonium in 2019 to analyze nitrification rate in the hypolimnion of Lake Constance. The bundled publication also includes measurements of chlorophyll a concentrations in the upper water column and measurements of temperature and oxygen in the whole water column.