This study describes microbial and chemical source tracking approaches for water pollution in rural and urban catchments. Culturable faecal indicator bacteria, represented by Escherichia coli, were quantified. Microbial source tracking (MST) using host-specific DNA markers was applied to identify the origins of faecal contamination. Chemical source tracking (CST) was conducted to determine contaminants of emerging concern (CEC) of human/anthropogenic origin, including pharmaceuticals and personal care products (PPCPs) and endocrine-disrupting chemicals (EDCs). In addition, the eutrophication-causing macronutrients nitrogen and phosphorus were studied. MST tests revealed both anthropogenic and zoogenic faecal origins, with a dominance of human sources in the urban stream; non-human/environmental sources were prevalent in the rural creek. CST analyses revealed a higher number of CECs in the urban stream than in the rural watercourse. Positive correlations between PPCPs and both E. coli and the human DNA marker were uncovered in the urban stream, while in the rural creek, PPCPs were only highly correlated with the anthropogenic marker. Interestingly, macronutrients were strongly associated with primary faecal pollution origins in both watercourses. This correlation pattern determines the main pollutant contributors (anthropogenic or zoogenic) to eutrophication.