The Baltimore Ecosystem Study stream biofilm bacterial community composition was obtained from 8 long-term sampling network sites in and near the Gwynns Falls watershed to examine how bacterial communities differ along an urban-rural gradient. Sampling was conducted at the same time as stream chemistry sampling on 18 June 2014 and 21 Oct 2014. Note: biofilm samples were taken about 50 meters east from the Carroll Park monitoring station, just under the I95 highway overpass, due to high water depth, high water flow, and lack of rock substrates for sampling. This dataset presents the number of sequences matching the taxonomic classifications in a reference database of 16S rRNA genes. See the full metadata record for detailed methods.