Legitimacy is regarded as one critical aspect of biodiversity management and nature conservation arrangements. Multi-level governance is claimed to pose several challenges to legitimacy. The aim of this paper is to review some legitimacy challenges in multilevel governance contexts, and to analyse empirically biodiversity governance in different EU countries in the light of these challenges. Four legitimacy criteria - legal compatibility, accountability, representation and inclusion, and transparency - serve as a framework for theoretical and empirical analysis. The analysis is based on twelve cases of multilevel biodiversity governance from different EU countries. The results show that several of the legitimacy challenges in multilevel governance can be observed in the cases, for example the poor inclusion of certain concerns at some time points of the decision process, difficulties in being accountable towards multiple levels simultaneously, or the weak visibility of the decision process either for the general public or for the immediate participants.