Abstract Background Acute myeloid leukemia (AML) is associated with high rates of resistance to standard therapies, necessitating the exploration of novel treatment strategies. Venetoclax (VEN) has shown efficacy in AML, yet drug resistance remains a significant challenge. This study aims to explore the synergistic effects of combining dihydroartemisinin (DHA) with VEN to improve therapeutic outcomes in AML. Methods AML cell lines and primary cells from AML patients were treated with various concentrations of DHA, VEN and their combined regimen. The cytotoxic effects were evaluated using MTS assays, flow cytometry for apoptosis analysis, and cell cycle assessments. Protein levels of caspase-3, PARP, MCL-1, BCL-XL and C-MYC were analyzed to elucidate the underlying mechanisms of the observed synergy. Results The combination of VEN and DHA demonstrated a significant synergistic cytotoxic effect on AML cells, characterized by reduced cell proliferation, induced apoptosis, and cell cycle arrest in the G0/G1 phase. Mechanistically, the synergy was associated with increased levels of cleaved caspase-3 and PARP, along with the downregulation of anti-apoptotic proteins MCL-1 and BCL-XL. Additionally, the combined treatment led to a significant decrease in C-MYC expression. This synergistic effect was consistently observed across all primary AML patient samples analyzed. Conclusion The findings suggest that the combination of VEN and DHA exerts synergistic anti-leukemic effects by targeting BCL-XL, MCL-1 and C-MYC, offering a promising therapeutic strategy for AML.