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Abstract Evaluating the impacts on biodiversity by means of the life cycle assessment (LCA) is a task researchers are currently facing. One impact assessment method that aims to do so is the recently developed biodiversity impact assessment BIA+. The aim of the present paper is to test this method and to show its applicability and feasibility. Additionally opportunities and challenges of this method shall be revealed. For this purpose, a case study for assessing the impacts of biodiesel production on freshwater biodiversity is introduced. Two substances, namely phosphorus and 1,4-dichlorobenzene (DCB), which are used for the biodiesel production for fertilization and the usage of pesticides, are being considered. The inventory data needed for the case study is gathered through the grey water footprint of the crop production that forms the basis of the European biodiesel consumption. Further, the BIA+ method is applied in order to assess the impacts of the biodiesel consumption in Europe for the year 2010 on freshwater biodiversity. The results reveal that even though assumptions have to be made and the results are highly uncertain, this method helps to identify hotspots within production processes. By including the BIA+ as an impact assessment method into LCA, impacts of products can be assessed with regard to biodiversity including genetic, species and ecosystem diversity. The main impact hotspot occurs in France with a potential loss of the biodiversity of 0.5 points. The lowest loss of the biodiversity occurs in Brazil (loss of 8.05*10–06 points). The results are mainly influenced by the impact of the toxic substance 1,4-DCB. By means of the case study, it is shown that the BIA+ method is applicable. [ABSTRACT FROM AUTHOR]