Psoraleae　Fructus　(PF)　is　a　widely-used　herb　with　diverse　pharmacological　activities,　while　its　related　hepatic　injuries　have　aroused　public　concerns.　In　this　work,　a　systematic　approach　based　on　RNA　sequencing　(RNA-seq),　high-content　screening　(HCS)　and　molecular　docking　was　developed　to　investigate　the　potential　mechanism　and　identify　major　phytochemicals　contributed　to　PF-induced　hepatotoxicity.　Animal　experiments　proved　oral　administration　of　PF　water　extracts　disturbed　lipid　metabolism　and　promoted　hepatic　injuries　by　suppressing　fatty　acid　and　cholesterol　catabolism.　RNA-seq　combined　with　KEGG　enrichment　analysis　identified　mitochondrial　oxidative　phosphorylation　(OXPHOS)　as　the　potential　key　pathway.　Further　experiments　validated　PF　caused　mitochondrial　structure　damage,　mtDNA　depletion　and　inhibited　expressions　of　genes　engaged　in　OXPHOS.　By　detecting　mitochondrial　membrane　potential　and　mitochondrial　superoxide,　HCS　identified　bavachin,　isobavachalcone,　bakuchiol　and　psoralidin　as　most　potent　mitotoxic　compounds　in　PF.　Moreover,　molecular　docking　confirmed　the　potential　binding　patterns　and　strong　binding　affinity　of　the　critical　compounds　with　mitochondrial　respiratory　complex.　This　study　unveiled　the　underlying　mechanism　and　phytochemicals　in　PF-induced　liver　injuries　from　the　view　of　mitochondrial　dysfunction.　©　2024,　The　Author(s).