Freshwater estuaries throughout the Great Lakes region receive stormwater runoff and riverine inputs from heavily urbanized population centers. While human and animal feces contained in this runoff are often the focus of source tracking investigations, non-fecal bacterial loads from soil, aerosols, urban infrastructure, and other sources are also transported to estuaries and lakes. We quantified and characterized this non-fecal urban microbial component using bacterial 16S rRNA gene sequences from sewage, stormwater, rivers, harbor/estuary, and the lake surrounding Milwaukee, WI, USA. Bacterial communities from each of these environments had a distinctive composition, but some community members were shared among environments. We used a statistical biomarker discovery tool to identify the components of the microbial community that were most strongly associated with stormwater and sewage to describe an "urban microbial signature," and measured the presence and relative abundance of these organisms in the rivers, estuary, and lake. This urban signature increased in magnitude in the estuary and harbor with increasing rainfall levels, and was more apparent in lake samples with closest proximity to the Milwaukee estuary. The dominant bacterial taxa in the urban signature were Acinetobacter, Aeromonas, and Pseudomonas, which are organisms associated with pipe infrastructure and soil and not typically found in pelagic freshwater environments. These taxa were highly abundant in stormwater and sewage, but sewage also contained a high abundance of Arcobacter and Trichococcus that appeared in lower abundance in stormwater outfalls and in trace amounts in aquatic environments. Urban signature organisms comprised 1.7% of estuary and harbor communities under baseflow conditions, 3.5% after rain, and >10% after a combined sewer overflow. With predicted increases in urbanization across the Great Lakes, further alteration of freshwater communities is likely to occur with potential long term impacts on the function of estuarine and nearshore ecosystems.