We explore some of the key challenges and opportunities that lie in assessing the role of freshwater biodiversity in sustaining ecosystem services, using the recent large interdisciplinary NERC-DURESS project (www.nerc-DURESS.org) as an exemplar case study of wider issues. The conceptual and methodological challenges raised are identified, explored and a range of methods are proposed to quantify how freshwater ecoservices, such as fish production or water quality regulation, depend on river organisms, and how we might identify biodiversity thresholds under which a service is likely to be compromised. We conclude that interdisciplinary, large scale, in situ approaches like these are needed to (i) fully understand how river biodiversity sustains ecosystem services; (ii) help evaluate if, where, and how the ecosystem approach can benefit long-term resource management and (iii) maximise impacts on policy, practice and decision making, which can be especially effective where strong ‘co-production’ partnerships with a broad range of stakeholders are developed and nurtured from a project's outset.