Several beaches within the Toronto region area of concern have persistent issues with fecal contamination, causing a beach beneficial use impairment (BUI). In this study, Escherichia coli, including ampicillin-resistant strains, were enumerated via culturable and quantitative polymerase chain reaction (qPCR) methods. Microbial source tracking (MST) markers (for general Bacteroidales, human, ruminant/cow, gull, and dog) were detected and enumerated via PCR and qPCR to identify sources of fecal contamination at Sunnyside Beach and in the Humber River. Human, cow, and dog markers had good host-specificity, while gull markers sometimes amplified a few other bird species. The ruminant endpoint PCR marker amplified a variety of other animal species rendering it less useful. Both human and gull fecal contamination were prevalent in the Humber River, while Sunnyside Beach was predominantly impacted by gull fecal contamination. Human sewage impacts were more prevalent in the lower Humber River, particularly in Black Creek. However, to reduce Sunnyside beach postings, reducing bird fecal contamination in the river and at the beach would be necessary. When there are high levels of E. coli throughout a beachshed, an MST toolbox approach can add value to discriminate source(s) of E. coli contamination and guide decisions relating to public health risk and remediation strategies.