We evaluated the biological water quality in relation to chemicals discharged through sewage outfall during both dry and rainy season. The lowland area of the Cuenca River basin in the southern Andes of Ecuador, including the city of Cuenca, constituted the study area. To perform an integrated water quality assessment, data were collected of macroinvertebrates, physicochemical conditions and morphological characteristics in 43 sites in the Cuenca River and its tributaries. The Andean Biotic Index (ABI) and the Biological Monitoring Working Party adapted to Colombia (BMWP-Col) were used to evaluate the biological water quality. Both biological indexes were higher upstream than downstream from the city. Moreover, these indexes indicated better conditions during the rainy season than in the dry season, based on the presence of more sensitive families. The biological indexes related more to the oxygen saturation than to the five-day biological oxygen demand (BOD5), nutrients and chloride concentrations. The relationship between BOD5 and nutrient concentrations with the variation of both biological indexes was clearer in the dry season than in the rainy season. However, in some sites, these indexes were influenced more by morphological aspects than by pollutants. Both biological indexes showed similar patterns along the rivers, generally the BMWP-Col scored higher than the ABI index. The latter index was shown to be more suitable for the high Andes region. These results could be used to support the implementation of river restoration actions, such as determining priorities for splitting sewer and precipitation water transport systems and needs for improved wastewater treatment facilities in specific locations.