Drinking water contamination poses ongoing public health concerns due to limited analytical and toxicological coverage of emerging and unknown chemical contaminants. Here, we integrate suspect screening and nontarget screening with in vivo toxicity testing to characterize organic chemical contaminants in tap drinking water across western Oregon, USA. Twelve public tap drinking water samples and one commercially bottled water sample were analyzed using complementary extraction and chromatographic workflows paired with high-resolution mass spectrometry analysis. Suspect screening identified 52 Level 1-2 annotated compounds, with prioritization leading to 13 chemical confirmations including both known drinking water contaminants (e.g., propiconazole, benzyl butyl phthalate, saccharin) and previously unreported drinking water contaminants (e.g., 18β-glycyrrhetinic acid). Zebrafish embryo and larvae toxicity screening demonstrated significant morphological toxicity following exposure to 18β-glycyrrhetinic acid and a benzyl butyl phthalate/monobenzyl phthalate mixture, while behavioral assays identified altered behavior following exposure to one individual contaminant and seven mixtures. This integrated analytical-toxicological framework demonstrates broad physicochemical screening, expands the inventory of confirmed drinking water contaminants, and establishes primary reference concentration values at the 20% effect level for one novel drinking water contaminant and several representative contaminant mixtures.