Aquatic ecological surveys are valuable to understanding the interaction between the biotic and abiotic components in rivers and streams. However, large-scale assessments of the water chemistry, geomorphology, and ecological community are usually not feasible due to limited resources. Beginning in 2013, the Regional Stream Quality Assessment Project of the US Geological Survey’s National Water Quality Program, began sampling 89-120 streams in each of 5 regions across the conterminous United States—the Midwest (2013), Southeast (2014), Pacific Northwest (2015), Northeast (2016), and California (2017). Sampling included water and streambed sediment chemistry, stage and temperature (Journey and others, 2015). The abiotic data is available from the National Water Information System (nwis.waterdata.usgs.gov). Geospatial data for the Southeastern U.S. study sites are available from Qi and others (2017). Ecological data collected included benthic algae, macroinvertebrates, and fish communities, in addition to in-stream habitat and geomorphology measurements for each reach. Ecological and habitat data for the Southeastern United States are summarized in this data release.