A series of 5-substituted 2-cyanoimino-4-imidazodinone and 2-cyanoimino-4-pyrimidinone derivatives were synthesized and their anticancer cytotoxicity were evaluated in in vitro assay. It was found that the bulky aryl functionality in the 5-position of the 2-cyanoimino-4-imidazolidinone compounds was essential for the cytotoxicity of these heterocyclic compounds. Some of the derivatives exhibited modest cytotoxicity against a variety of cancer cell lines. One of the derivatives, [1-[6-(4-chlorophenoxy)hexyl]-5-oxo-4-phenyl-3-(4-pyridyl)tetrahydro-1H-2-imidazolyliden]aminomethanenitrile (Compound 11), exhibited the most potent cytotoxic activity with IC(50) in the nanomolar range. The cytotoxicity of these derivatives was selection with no apparent toxic effect toward normal fibroblasts.