All-trans retinoic acid (ATRA) is a derivative of vitamin A and exhibits anticancer activity against acute promyelocytic leukemia. Fenbendazole (FBZ) is a benzimidazole anthelmintic that has wide safety margin and low toxicity. Recently, FBZ has been found to have anticancer activity by destabilizing microtubules. In this study, we treat ATRA and FBZ on HL-60 cells, a human leukemia cell line, to investigate the synergistic effects of two drugs, and the potential anticancer mechanism. ATRA and FBZ significantly decreased the metabolic activity of HL-60 cells at 0.04 μM ATRA. Cell viability of ATRA-treated HL-60 cells decreased in a concentration-dependent manner and more decreased by FBZ. N-acetyl cysteine, an inhibitor of reactive oxygen species production, significantly increased the metabolic activity of the cells treated with ATRA and FBZ. Hoechst 33342 and propidium iodide staining showed the presence of broken nuclei in the HL-60 cells treated with ATRA and FBZ. And also, an apoptosis analysis demonstrated that 0.2 μM FBZ increased the percentages of cells in apoptosis and necrosis. In contrast, 0.04 μM ATRA showed no significant difference. Based on multiple assays, ATRA and FBZ showed not synergistic, but additive effect on HL-60 cells. This study may provide researchers and clinicians in cancer-related fields with some valuable information regarding the application of ATRA and FBZ.