AIM: To investigate the effects of 7,8-dihydroxyflavone (DHF) on arsenic trioxide (ATO)-induced cardiomyocyte cytotoxicity and mitochondrial dysfunction, as well as the underlying mechanisms. METHODS: HL-1 and H9c2 cardiomyocyte cell lines were used to establish an ATO-induced cell injury model, followed by DHF intervention. Cell morphology was observed under a light microscope, cell viability was assessed using the MTT assay, and cell survival rate was evaluated via Calcein-AM staining. Mitochondrial respiratory function (including basal respiration, maximal respiration, and reserved respiratory capacity) was measured using a high-resolution mitochondrial respirometry system. The protein expression levels of AKT, HO-1, and Nrf2 were detected by Western blot. RESULTS: ATO (5, 10 μmol/L) induced damage of and impaired mitochondrial respiratory function in HL-1 cells. DHF intervention further exacerbated ATO-induced HL-1 cell damage and mitochondrial dysfunction. In H9c2 cells, ATO (5 μmol/L) inhibited survival and reduced cell viability, while DHF treatment aggravated these detrimental effects, suppressed mitochondrial respiration, which was associated with increased phosphorylated AKT and HO-1 protein expression, and decreased Nrf2 expression. CONCLUSION: DHF exacerbates ATO-induced cardiomyocyte cytotoxicity and mitochondrial respiratory dysfunction.