Perfluorodecanoic acid (PFDA), a long-chain perfluoroalkyl substance (PFAS), is known for its environmental persistence and potential toxicity. This study evaluated PFDA toxicity in the RTgill-W1 cell line, a model for aquatic toxicology, using a combination of cell viability assays, reactive oxygen species (ROS) measurements, and high-throughput metabolomics and lipidomics. PFDA exposure resulted in significant, dose-dependent reductions in cell viability and increased ROS production, with an EC₅₀ value of 51.9 ± 1.7 mg/L, highlighting its cytotoxic potential. Metabolomic profiling revealed dose-dependent disruptions in 168 metabolites, impacting pathways related to amino acid metabolism, carbohydrate metabolism, lipid metabolism, vitamin and cofactor metabolism, and nucleotide metabolism. Furthermore, lipidomic analysis identified 102 significantly altered lipids, primary affecting glycerolipid metabolism, fatty acid biosynthesis, glycerophospholipid metabolism, sphingolipid metabolism - suggesting compromised membrane integrity, energy production, and signalling processes. These findings underscore PFDA's capacity to interfere with critical cellular processes and highlight the utility of integrated omics approaches in elucidating the molecular mechanisms of PFAS toxicity. Future studies should focus on validating fish cell assays through short-term in vivo tests to enhance their reliability and ecological relevance.