Per- and polyfluoroalkyl substances (PFAS) are persistent endocrine-disrupting chemicals (EDCs) linked to adverse reproductive outcomes. While the placenta is a known target of PFAS toxicity, most in vitro studies use two-dimensional (2D) cell culture models, often focusing on late-pregnancy tissue or blood PFAS concentrations and examining single compound exposures. In this study, we measure placenta PFAS concentrations in early pregnancy and design a placenta-relevant PFAS mixture to assess its impact on trophoblast function using a three-dimensional (3D) trophoblast spheroid model. PFAS levels in first-trimester placental tissue were quantified using liquid chromatography/triple quadrupole mass spectrometry. Six PFAS: perfluorononanoic acid (PFNA), perfluorooctanesulfonic acid (PFOS), perfluorobutanoic acid (PFBA), perfluorooctanoic acid (PFOA), perfluorohexanesulfonic acid (PFHxS), and perfluorodecanoic acid (PFDA); were selected based on their placenta concentrations and relevance to pregnancy complications to design the placenta real-life PFAS mixture. Next, trophoblast spheroids were propagated from two different cell lines, JEG-3 and HTR-8/SVneo, to assess the effects of PFAS mixture on trophoblast viability, apoptosis, invasion, hormone production, and gene expression. While trophoblast spheroid viability remained largely unaffected, we observed changes in trophoblast function. PFAS exposure significantly increased invasiveness in JEG-3 spheroids at 48 h, but markedly reduced it in HTR-8/SVneo spheroids at 96 h across varying concentrations. Additionally, pregnancy-specific hormone e.g. β-hCG production declined after 48 h of PFAS mixture exposure in JEG-3 spheroids. Gene expression analysis revealed altered apoptosis and proliferation pathways in both trophoblast spheroids. Overall, our study highlights that physiologically relevant 3D trophoblast models can contribute to the broader comprehension of PFAS-associated reproductive health risk assessments.