Multiple microbial source-tracking methods were investigated to determine the source of elevated Escherichia coli levels at Bayfront Park Beach in Hamilton Harbour, Lake Ontario. E. coli concentrations were highest in wet foreshore sand (114,000 CFU/g dry sand) and ankle-depth water (177,000 CFU/100mL), declining rapidly in deeper waters. Many gull and geese droppings were enumerated each week on the foreshore sand within 2m of the waterline. Both antimicrobial resistance analysis and rep-PCR DNA fingerprinting of E. coli collected at the beach and nearby fecal pollution sources indicated that E. coli in sand and water samples were predominantly from bird droppings rather than from pet droppings or municipal wastewater. Both methods indicated a trend of decreasing bird contamination, and increasing wastewater contamination, moving offshore from the beach. When foreshore sand was treated as a reservoir and secondary source of E. coli, waterborne E. coli were found to be more similar to sand isolates than bird or wastewater isolates out to 150 m offshore. Multiple lines of evidence indicated the importance of bird droppings and foreshore sand as primary and secondary sources of E. coli contamination in beach water at Bayfront Park.