The Guayas river basin is one of the major watersheds in Ecuador, where increasing human activitiesare affecting water quality and related ecosystem services. The aims of this study were (1) to assess theecological water quality based on macroinvertebrate indices and (2) to determine the major environ-mental variables affecting these macroinvertebrate indices. To do so, we performed an integrated waterquality assessment at 120 locations within the river basin. Biological and physical–chemical data werecollected to analyze the water quality. Two biotic indices were calculated to assess the water qualitywith an ecological approach: the Biological Monitoring Working Party Colombia (BMWP-Col) and theNeotropical Low-land Stream Multimetric Index (NLSMI). Both the BMWP-Col and NLSMI indicated goodwater quality at the (upstream) forested locations, lower water quality for sites situated at arable landand bad water quality at residential areas. Both indices gave relevant assessment outcomes and can beconsidered valuable for supporting the local water management. A correspondence analysis (CA) appliedon both indices suggested that flow velocity, chlorophyll concentration, conductivity, land use, sludgelayer and sediment type were the major environmental variables determining the ecological water qual-ity. We also suggested that nutrient and pesticide measurements are important to study water qualityin the area where intensive agriculture activities take place. The nutrient levels detected in agriculturalareas were relatively low and illustrated that the types of crops and the current cultivation methods werenot leading to eutrophication. The applied methods and results of this study can be used to support thefuture water management of the Guayas river basin and similar basins situated in the tropics.