Tire wear-derived toxic organic compounds (TWTOCs) are emerging environmental pollutants generated from vehicle tire abrasion, yet their cellular toxicity mechanisms remain poorly understood. In this study, human lung epithelial cells A549 and human hepatocellular carcinoma cells Hep3B were used to investigate the toxicological effects of three TWTOCs: N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), 1,3-diphenylguanidine (DPG), and N-isopropyl-N-phenyl-4-phenylenediamine (IPPD). First, the physicochemical properties and potential multi-organ toxicities of 6PPD, DPG, and IPPD were predicted using ADMETlab 3.0. Cytotoxicity was assessed using the CCK-8 assay, followed by metabolomic and lipidomic analyses after 24 h exposure. TWTOCs exposure significantly reduced cell viability and induced pronounced metabolic perturbations, particularly in amino acid metabolism, folate-mediated one-carbon metabolism, and pantothenate–CoA biosynthesis pathways. Lipidomic analysis revealed notable alterations in arachidonic acid–containing lipid species. In addition, inflammatory cytokine analysis by ELISA demonstrated increased IL-6 and TNF-α levels and decreased IL-10. These findings indicate that TWTOCs may trigger oxidative stress and inflammatory responses by disrupting key metabolic pathways, thereby exerting cytotoxic effects. This study provides mechanistic insights into TWTOC toxicity and highlights their potential environmental health risks.