Reference conditions for river bioassessment should be established inside ecoregions. Our objectives were (1) to implement a bioassessment methodology for Uruguayan prairie streams regarding ecoregions and land use and (2) to assess the ecological integrity of streams of the Río Negro basin in Uruguay. Due to logistical constraints, sampling was divided into two collection trips: one including the upper basin in fall 2015 and the other including the lower basin in spring 2016. Basins were analyzed separately due to seasonal and geographical differences. In the streams sampled in fall 2015, conductivity, total nitrogen (TN), and total phosphorus (TP) were higher in sedimentary ecoregions than in crystalline ones, independent on land use. In those sampled in spring 2016, these variables showed the highest values in the ecoregions dominated by agriculture. Eighty percent of the sampled streams presented the impact of cattle in their riparian zone. Discriminant analysis showed a similar composition of macroinvertebrates among ecoregions in 2015, but different composition between land uses. Conversely, in 2016, there were differences among some ecoregions, but not between land uses. Agriculture was correlated with tolerant invertebrates, while natural land use and afforestation were correlated with sensitive ones. The BMWP-Colombia showed the impact of livestock on streams, but in general good water quality, while an index of genera for Uruguay, indicated that all streams are eutrophic, thereby confirming the importance of using different types of metrics. Due to its geographical homogeneity and small size, a smaller number of ecoregions could be defined for stream assessments in the Río Negro basin. [ABSTRACT FROM AUTHOR]