In order to develop novel anti-cancer agents, a series of asymmetrical 2,6-bis (benzylidene)cyclohexanone derivatives containing nitrobenzylidene moiety were synthesized and their cytotoxic activity were determined in vitro against MDA-MB 231, MCF-7 and SK-N-MC cell lines using MTT assay. Among the tested compounds, the highest activity against MDA-MB 231 cells was achieved by 2-(3-bromo-5-methoxy-4-propoxybenzylidene)-6-(2-nitrobenzylidene)cyclohexanone (compound 5d). Whereas, compound 5j (the 3-nitro analog of compound 5d) was the most potent compound against MCF-7 and SK-N-MC cell lines. The results indicated that the cytotoxic activity profile against different tumor cells can be optimized by desired 4-alkoxy-3-bromo-5-methoxybenzylidene scaffold.