Sustainability assessment (SA) is a holistic and long-range strategic instrument capable of assisting policy-making in electing, and deciding upon, future development priorities. The outcomes of an SA process become more relevant and strengthened when conducted with multi-stakeholder engagement, which provides for multiple dialogues and perspectives. This was the object of research of the SA team in the context of BioScene (Scenarios for Reconciling Biodiversity Conservation with Declining Agriculture Use in Mountain Areas in Europe), a three-year project (2002-2005) funded by the European Union 5th Framework Program, which aimed to investigate the implications of agricultural restructuring and decline for biodiversity conservation in the mountain areas of Europe, using three distinct methodological streams: the ecological, the socio-economic, and the SA approaches. The SA approach drew on the previous two to assess the importance for biodiversity management of different scenarios of agri-environmental change and rural policy in six countries (France, Greece, Norway, Slovakia, Switzerland, and the United Kingdom), develop causal chains, include stakeholder views, and identify potential contributions for, or conflicts with, sustainability. This article tells how SA was used, what sustainability meant in each study area through different objectives of sustainability considered, discusses the methods used in SA, and the benefits arising. The SA was conducted by a team independent of any study area, who developed and oversaw the application of the SA methodology, assisting national teams, and developing a cross-country understanding of the sustainability of proposed scenarios in the different geographical and social contexts, and their implications for policy-making. Finally, it reflects on the persistent challenges of interdisciplinary research, compounded by multi-cultural teams, and concludes on the BioScene's lessons for the further development and application of SA.