The horizontal, fluorophore enhanced, rep-PCR (HFERP) DNA fingerprinting technique was used to identify potential sources of Escherichia coli in water, nearshore sand, and sediment at two beaches in the Duluth-Superior Harbor, near Duluth, MN, and Superior, WI, during May, July, and September 2006. An animal or environmental source could be identified for 35, 29, and 30% of E. coli strains in water, sand, and sediments, respectively. Waterfowl, including Canada geese, ring-billed gulls, and mallard ducks, were the largest source of E. coli that could be identified in water (55–100%), sand (59–100%), and sediment (92–100%) at both beaches. Although ring-billed gulls were more abundant in this harbor, Canada geese were usually the dominant source of waterfowl E. coli found at these beaches. The percentage of E. coli identified from treated wastewater was always less than the percentage of E. coli originating from waterfowl. At both beaches, the percentage of E. coli in water contributed by treated wastewater was higher in May compared with July and September. The larger proportion of wastewater-derived E. coli seen in May probably reflected a smaller contribution of E. coli from geese when these birds were less abundant rather than an absolute increase in E. coli from treated wastewater. Microbial source analysis and bird census data both indicated that waterfowl were a major source of E. coli at beaches in the Duluth-Superior Harbor. These data also indicated it is risky to assume that the most abundant waterfowl species present in waterways will also be the largest source of avian-derived E. coli in water, nearshore sand, and sediments at beaches.