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In Germany, 70 % of the drinking water demand is met by groundwater, for which the quality is the product of multiple physical–chemical and biological processes. As healthy groundwater ecosystems help to provide clean drinking water, it is necessary to assess their ecological conditions. This is particularly true for densely populated urban areas, where faunistic groundwater investigations are still scarce. The aim of this study is, therefore, to provide a first assessment of the groundwater fauna in an urban area. Thus, we examine the ecological status of an anthropogenically influenced aquifer by analysing fauna in 39 groundwater monitoring wells in the city of Karlsruhe (Germany). For classification, we apply the groundwater ecosystem status index (GESI), in which a threshold of more than 70 % of crustaceans and less than 20 % of oligochaetes serves as an indication for very good and good ecological conditions. Our study reveals that only 35 % of the wells in the residential, commercial and industrial areas and 50 % of wells in the forested area fulfil these criteria. However, the study did not find clear spatial patterns with respect to land use and other anthropogenic impacts, in particular with respect to groundwater temperature. Nevertheless, there are noticeable differences in the spatial distribution of species in combination with abiotic groundwater characteristics in groundwater of the different areas of the city, which indicate that a more comprehensive assessment is required to evaluate the groundwater ecological status in more detail. In particular, more indicators, such as groundwater temperature, indicator species, delineation of site-specific characteristics and natural reference conditions should be considered.