The complexity of contaminant mixtures in surface waters has presented long-standing challenges to the assessment of risks to human health and the environment. As a result, novel strategies for both identifying contaminants that have not been routinely monitored through targeted methods and prioritizing detected compounds with respect to their biological relevance are needed. Tracking biotransformation products in biofluids and tissues in an untargeted fashion facilitates the identification of chemicals taken up by the resident species (e.g., fish), so by default ensuring that detected compounds are biologically relevant in terms of exposure. In this study, we investigated xenobiotic glucuronidation, which is arguably the most important phase II metabolism pathway for many pharmaceuticals, pesticides, and other environmental contaminants. The application of an untargeted high-resolution mass spectrometry-based approach tentatively revealed the presence of over 70 biologically relevant xenobiotics in bile collected from male and female fathead minnows exposed to wastewater treatment plant effluents. The majority of these were not targets of conventional contaminant monitoring. These results highlight the utility of biologically based untargeted screening methods when evaluating chemical contaminants in complex environmental mixtures.