This study assessed the water quality and ecological integrity of the Columbe River micro-watershed in the Ecuadorian Andes through a multidimensional approach incorporating biotic, physicochemical, and structural indices. Indices such as the Andean Biotic Index (ABI), Biological Monitoring Working Party index adapted for Colombian conditions (BMWP-Col), Fluvial Habitat Index (IHF), Riparian Quality Index adapted for Andean conditions (QBR-And), and Water Quality Index (WQI) characterized environmental quality gradients and evaluated the impact of human activities across 11 monitoring sites. Hierarchical cluster analysis classified sampling sites into three groups: less polluted (LP), moderately polluted (MP), and highly polluted (HP). HP sites showed elevated levels of biochemical oxygen demand (BOD5), chemical oxygen demand (COD), electrical conductivity (EC), and turbidity, alongside low biotic and structural scores, indicating advanced ecological degradation. Conversely, LP sites demonstrated greater ecological integrity, despite elevated BOD5 and COD levels across the watershed, suggesting widespread diffuse contamination. The findings identify anthropogenic activities such as livestock, agriculture, and domestic discharges as major pressures on water quality and macroinvertebrate biodiversity. Significant correlations between physicochemical parameters—including BOD5 and EC—and declining biotic indices underscore the link between chemical water degradation and ecological fragmentation. In this context, this study highlights the critical need for comprehensive management and restoration strategies to combat pollution, safeguard relatively pristine areas, and rehabilitate the ecological integrity and connectivity of high-altitude Andean aquatic ecosystems under anthropogenic pressure.