High-resolution mass spectrometry is advantageous for monitoring physiological impacts and contaminant biotransformation products in fish exposed to complex wastewater effluent. We evaluated this technique using skin mucus from male and female fathead minnows (Pimephales promelas) exposed to control water or treated wastewater effluent at 5%, 20%, and 100% levels for 21 d, using an onsite, flow-through system providing real-time exposure. Both sex-specific and non-sex-specific responses were observed in the mucus metabolome, the latter suggesting the induction of general compensatory pathways for xenobiotic exposures. Altogether, 85 statistically significant treatment-dependent metabolite changes were observed and 30 of those annotated with probable structures. The mummichog software package was used to elucidate impacted biochemical pathways and enhance metabolite annotation. Partial least squares regression models revealed relationships between the mucus metabolomes and upregulated hepatic mRNA transcripts reported previously for these same fish. These regression models suggest that mucus metabolomic changes reflected, in part, processes by which the fish biotransformed xenobiotics in the effluent. Further, we detected a phase II transformation product of bisphenol A in the skin mucus of male fish. Collectively, these findings demonstrate the utility of mucus as a minimally invasive matrix for simultaneously assessing exposures and effects of real-world mixtures of contaminants. This dataset is associated with the following publication: Mosley, J., D. Ekman, J.E. Cavallin, D. Villeneuve, G. Ankley, and T. Collette. High‐resolution mass spectrometry of skin mucus for monitoring physiological impacts and contaminant biotransformation products in fathead minnows exposed to wastewater effluent. ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY. Society of Environmental Toxicology and Chemistry, Pensacola, FL, USA, 37(3): 788-796, (2018).