CLL remains an incurable disease, making it crucial to continue searching for new therapies efficient in all CLL cases. We have studied the effect of combining arsenic trioxide (ATO) with fludarabine, a frontline drug in CLL. We have found a synergistic interaction between 1 μM ATO and 5 μM fludarabine that significantly enhanced the cytotoxic effect of the individual drugs. Importantly, ATO sensitized fludarabine-resistant cells to the action of this drug. The mechanism behind this effect included the downregulation of phospho-Akt, phospho-ERK, and the Mcl-1/Bim and Bcl-2/Bax ratios. The combination of ATO and fludarabine partially overcame the survival effect induced by co-culturing CLL cells with stromal cells. Therefore, low concentrations of ATO combined with fludarabine may be an efficient therapeutic strategy in CLL patients. •We have identified a synergistic interaction between ATO and fludarabine.•ATO sensitize to fludarabine the CLL cases with poor response to this drug.•The combination ATO/fludarabine downregulates crucial survival pathways in CLL.•The combination of ATO/fludarabine partially overcomes the survival effect of stroma.•ATO may be efficient for CLL treatment in combined therapies with fludarabine.