Multiple microbial source tracking methods were applied to investigate spatial variation in faecal pollution sources impacting a 1.7 km freshwater beach on Lake Ontario (Canada). The highest E. coli concentrations measured in the study area were from interstitial sand pore water at Sunnyside Beach, reaching 2.6 × 106 CFU/100 ml. These E. coli concentrations exceeded those in the nearby Humber River and Black Creek, which are impacted by combined sewer overflows containing municipal wastewater and by stormwater conveying washoff from the urban area. Library-independent Bacteroidales HF183 analyses identified the more frequent occurrence of municipal wastewater contamination in the Humber River and at a Sunnyside Beach location closest to the mouth of the river. Library-dependent E. coli antibiotic resistance and rep-PCR DNA fingerprinting analyses identified the more frequent occurrence of bird faecal contamination at Sunnyside Beach locations away from the river mouth. These microbial source tracking results raise caution about managing beaches with multiple sources of contamination as a single entity without considering spatial variability in faecal pollution sources and the need for more localized beach management practices.