Biodiversity loss is caused by intensive human activities and threatens human well-being. However, less is known about how the combined effects of multiple stressors on the diversity of internal (alpha diversity) and multidimensional (beta diversity) communities. Here, we conducted a long-term experiment to quantify the contribution of environmental stressors (including water quality, land use, climate factors, and hydrological regimes) to macroinvertebrate communities alpha and beta diversity in the mainstream of the Songhua River, the third largest river in China, from 2012 to 2019. Our results demonstrated that the alpha and beta diversity indices showed a decline during the study period, with the dissimilarity in community composition between sites decreasing significantly, especially in the impacted river sections (upper and midstream). Despite overall improvement in water quality after management intervention, multiple human-caused stressors still have led to biotic homogenization of macroinvertebrate communities in terms of both taxonomic and functional diversities in the past decade. Our study revealed the increased human land use explained an important portion of the variation of diversities, further indirectly promoting biotic homogenization by changing the physical and chemical factors of water quality, ultimately altering assemblage ecological processes. Furthermore, the facets of diversity have distinct response mechanisms to stressors, providing complementary information from the perspective of taxonomy and function to better reflect the ecological changes of communities. Environmental filtering determined taxonomic beta diversity, and functional beta diversity was driven by the joint efforts of stressors and spatial processes. Finally, we proposed that traditional water quality monitoring alone cannot fully reveal the status of river ecological environment protection, and more importantly, we should explore the continuous changes in biodiversity over the long term. Meanwhile, our results also highlight timely control of nutrient input and unreasonable expansion of land use can better curb the ecological degradation of rivers and promote the healthy and sustainable development of floodplain ecosystems.

High‐throughput pipelines supported by eDNA metabarcoding have been applied in various freshwater ecosystems. Both eDNA in ethanol (EtOH) samples (ES‐eDNA) and in water samples (WS‐eDNA) can provide comprehensive classification lists with good taxonomic resolution and coverage for determining freshwater biodiversity and biomonitoring. But, the advantages of ES‐eDNA metabarcoding over WS‐eDNA metabarcoding remain unclear for routine assessments of diversity of benthic macroinvertebrates in streams.