Rivers in the Andean region are subject to growing anthropogenic and climatic pressures that compromise water quality and biodiversity. This study assessed the physicochemical, microbiological, and toxicological characteristics of the Tahuando River (Ecuador) through integrated multi-bioassay approach, using seven sampling points along its urban and rural course during July 2023. Physicochemical parameters (pH, conductivity, dissolved oxygen, temperature), oils and fats (APHA 5520-B), and bioassays (Lactuca sativa germination, coliform counts, EBPI Toxi-ChromoTest) were evaluated. Conductivity increased significantly downstream (137.6 µS/cm to 669 µS/cm), while pH shifted toward alkaline values (7.72 to 8.14). Oils and fats remained below regulatory limits, peaking at 0.24 mg/L and 0.22 mg/L at Points 6 and 7. Lactuca sativa germination indicated low to moderate toxicity, with the lowest normalized germination index at Point 5. Microbiological results revealed E. coli proliferation at Points 6 and 7, exceeding acceptable thresholds. EBPI assays demonstrated toxicity values surpassing 20% MIC at Points 2, 6, and 7, indicating risks to aquatic microbiota. These findings identify three critical intervention points requiring riparian buffer zone restoration, wastewater treatment infrastructure, and livestock activity regulation to protect downstream communities and ecosystem health.