The Western Amazon is a global biodiversity hotspot, yet the Upper Napo River Basin (UNRB) remains understudied regarding aquatic ecosystem health along anthropogenic gradients. We integrated benthic macroinvertebrate assemblages with physicochemical and microbiological indicators across 45 sites to assess ecological quality under four impact scenarios: Few Threats (FT, reference sites; n = 6), Crop/Aquaculture (CA; n = 22), Gold Mining (GM; n = 10), and Wastewater Discharge (WD; n = 7). Analysis of 2285 individuals (62 families) revealed clear degradation across the anthropogenic gradient. Reference sites (FT) exhibited high integrity (q₀ = 24.3 families), establishing the regional baseline for Andean–Amazonian freshwater ecosystems. In stark contrast, GM sites showed catastrophic defaunation (q₀ = 9.9 families) coupled with extreme turbidity (1320 ± 1589 NTU) and heavy metal mobilization (Fe: 430 ± 229 µg/L; Cu: 338 ± 128 µg/L), placing these reaches in “Bad” ecological status (Ecological Quality Ratio, EQR ≤ 0.16). Wastewater sites reached critical fecal coliform levels (33,708 ± 58,047 CFU/100 mL)—165-fold higher than FT sites—indicating severe sanitary impairment and community collapse (EQR = 0.28, dominated by Chironomidae at 80%). The application of ASPT (Average Score Per Taxon) and EQR proved essential for detecting functional shifts toward tolerant assemblages even when raw biotic scores appeared moderate. Crop/Aquaculture sites showed intermediate degradation (EQR = 0.37–0.38), reflecting chronic pesticide exposure and habitat loss. We conclude that gold mining and wastewater discharge are the primary drivers pushing the UNRB toward ecological collapse, with GM exerting the most severe impact on aquatic biodiversity. Safeguarding this global freshwater stronghold requires immediate implementation of multimetric biomonitoring, enhanced mining regulation, wastewater treatment infrastructure, and establishment of Indigenous-led fluvial reserves to maintain long-term connectivity.