Benzyl-o-vanillin and benzimidazole nucleus serves as an important pharmacophore in drug discovery as it have a significant medicinal importance. Thus we explored the anticancer activity of three relative compounds and found their structure activity relationship compare with 2-benzyloxy-3-methoxybenzaldehyde main structure. According to the cytotoxicity analysis, they induce cell death of leukemic cell line through DNA fragmentation. DNA binding studies reveals that the DNA binding occurs through groove region. The higher mutagenicity might cause DNA mutation through interaction with the guanines while the lack of significant mutagenic activity suggests that the compound may target mainly the AT rich sequences of the nucleic acid. Hence, the present study provides a new insight of these novel (2-benzyloxy-3-methoxyphenyl) derivatives serving as a potential therapeutic agent against leukemia. This work should help shed some light on these exciting compounds and their SAR results, and should be especially useful to professionals in Oncology and cancer research feilds, or anyone else who may be considering utilizing DNA-drug interactions along with mutagenicity for research effort.