The assessment and monitoring of the ecological quality and status of groundwater is a timely issue. At present, various assessment tools have been developed that now await application and validation. One of these, the D‑A‑C index, evaluates the microbiological-ecological quality of groundwater based on of prokaryotic cell counts, microbial activity measurements, and the qualitative characterization of dissolved organic carbon (DOM). The purpose of this paper is to illustrate the different ways of application of the D‑A-(C) index making use of a recently collected data set (n = 61) from the river Mur valley, Austria. First, we present an extension of the D‑A-(C) index by including measurements of dissolved organic matter quality (DOM) derived from fluorescence spectroscopy as additional variables to supplement the analysis of microbial cell density and activity levels. Second, we illustrate how the definition of a reference status for a ‘good’ microbiological-ecological state can improve the analysis and allow for a more sensitive and accurate detection of impacts on groundwater ecosystems. Based on our results, we advocate that the analysis be performed by making use of expert knowledge for the definition of reference sites to which target sites are to be compared.