These data were used for an analysis as part of a long-term project monitoring the biological communities, habitat, and water quality of the Fountain Creek Basin. Biology, habitat, and water quality data have been collected at 10 sites starting in 2003. These data include annual samples of aquatic invertebrate communities, fish communities, water quality, and quantitative riverine habitat collected from 2003-2016. These data will provide insight into trends in biological communities from 2003-2016 and relationships between biological communities and abiotic variables (antecedent streamflow, physical habitat, and water quality). This USGS data release contains all the input and output data and files necessary to reproduce results of the report describing an analysis of ecological trends from 2003-2016 in the Fountain Creek Basin, Colorado, USA (https://doi.org/10.3133/sir20175162). Data preparation of these data files is fully described in the report mentioned previously. These data are used for a two-step analysis of trends in biological communities and the abiotic drivers of these trends. All files with a _1 suffix are used for the analysis of streamflow drivers of trends in these biological communities. All files with a _2 suffix contain data from unexplained trends after the first analysis and are used for an analysis exploring if these trends are explained by water quality or habitat characteristics.