<p>One of the foremost challenges in contemporary ecology is understanding the rapid loss of animal diversity and biomass, known as defaunation. The causes and consequences of defaunation depend on species traits that regulate which species are vulnerable to stressors and the ecosystem-level consequences of declines. We used a &gt; 30-year long-term monitoring dataset collected from 21 sites in 4 rivers in south-eastern Oklahoma, USA, and a trait-based framework to identify defaunation in multi-species freshwater animal aggregations (freshwater mussels). We detected mussel density declines in 2 of the 4 rivers, encompassing 67% of the study sites. In the defaunated rivers, losses of mussel density led to biomass declines and losses of mussel-generated carbon and nutrient recycling and storage. Mussel aggregations only became defaunated in rivers where hydrologic data suggested drought-like conditions. There was also a regional increase in temperature, suggesting that low flows and high temperatures may have negatively impacted mussel populations in the defaunated rivers. Declines did not differ among thermal tolerance guilds—rather, declines were driven by species with life-history traits adapted for stable hydrologic conditions. Given the significant role that river ecosystems play in global biogeochemical processes, freshwater defaunation may have major consequences if continued unchecked. More broadly, trait-based approaches show clear promise for advancing the predictive capacity of animal ecology.</p>