Background <p>Amniotic fluid (AF) provides a direct window into the prenatal exposome, yet it remains understudied despite the heightened vulnerability of the developing fetus to chemical and biological perturbations. Characterizing both endogenous metabolites and exogenous contaminants in AF is essential for understanding fetal chemical exposures.</p> Objective <p>To profile endogenous and exogenous chemical features in second-trimester AF and assess potential interactions between environmental contaminants and fetal metabolic pathways.</p> Methods <p>Non-targeted analysis (NTA) using liquid chromatography–high-resolution mass spectrometry (LC-HRMS) was applied to AF samples (<i>n</i> = 40) collected from participants in the San Francisco Bay Area. Compound annotation involved library matching and evaluation of diagnostic fragments. Correlation network analysis was performed to identify associations between exogenous chemicals and endogenous metabolites. Kendrick mass defect (KMD) analysis was used to distinguish chemical classes and prioritize unknown features for structural interpretation.</p> Results <p>A total of 290 compounds were annotated, including ten confirmed environmental contaminants. Nine of these ten contaminants, i.e., 1,3-diphenylguanidine, N,N-dimethyltetradecylamine-N-oxide, quinoline, phthalic anhydride, acetyl tributyl citrate, 4-hydroxybenzenesulfonic acid, lauryl sulfate, 4-dodecylbenzenesulfonic acid, and dibutyl adipate, are reported in AF for the first time. These chemicals spanned surfactants, plasticizers, and industrial intermediates, indicating diverse anthropogenic sources. Correlation analysis revealed links between several contaminants (e.g., tetradecyl sulfate, phthalic anhydride, quinoline) and endogenous lipid metabolites, suggesting possible metabolic disruption. KMD analysis further differentiated chemical classes and showed that many unknown features exhibited KMD signatures consistent with exogenous esters and amine/amide-containing compounds.</p> Significance <p>This study demonstrates the power of NTA to reveal previously undocumented prenatal exposures and uncover potential interactions between environmental chemicals and fetal metabolic processes. These findings expand our understanding of the prenatal exposome and highlight the need for continued investigation into environmental influences on fetal health.</p> <p></p> Impact <p>This study presents the first application of LC-HRMS non-targeted analysis to human amniotic fluid, uncovering nine environmental contaminants that have never previously been reported in this matrix. These newly detected surfactants, plasticizers, and industrial chemicals reveal a wider range of fetal exposures than currently recognized. By expanding the chemical landscape of the prenatal environment, this work highlights the importance of NTA for discovering emerging contaminants and underscores the need to evaluate their potential impacts on fetal development.</p>