There is a growing consensus that inappropriate valuation of the world's ecosystem services has historically led to widespread errors in environmental management, with associated negative social consequences. Freshwater ecosystems are prime examples: when managed appropriately, they provide major services, such as fish production, water supply, nutrient transport, health benefits and recreational value. However, these services are often compromised because they are seldom recognised explicitly in catchment use and planning. Moreover, pressures on river ecosystem services will grow as land use intensifies, water demands increase, and climate change accelerates over the coming decades. Maintaining and protecting river ecosystem services will depend increasingly on understanding the processes that underpin and degrade them, and especially in terms of characterising the roles played by the biota. While the integrity and stability of ecosystem processes tend to increase with biodiversity, how services and biodiversity are related is largely unknown, due to a range of unresolved practical and philosophical issues. 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.