The Centers for Disease Control and Prevention reported 6,939 deaths and 477,000 emergency department visits annually during 2009 to 2013 were attributed to 13 waterborne pathogens. The annual cost of hospitalization and treatment associated with these diseases totaled $3.8 billion. To combat water body impairment and limit the spread of disease, federal, state, and local agencies work to identify and remediate sources of microbial contamination in surface waters through microbial source tracking (MST) utilizing microbial and chemical tracers. The objective of this study is to identify novel chemical tracers of microbial contamination which could be utilized in MST efforts, furthering the ability of public health professionals to identify and remediate impaired waters. High-resolution mass spectrometry was used to analyze water samples from nine surface water sites within the Samish watershed which had been classified by a scent tracking canine regarding their presence of wastewater contamination. Chemicals unique to canine positive sites and those detected at a two-fold greater peak abundance in these sites were identified at varying confidence levels. A group of eight chemicals, including 2-mercaptobenzithizole, which were detected in three of five canine positive sites were identified as potential chemical tracers of wastewater.