Eight novel diamidino 3, 4-ethylenedioxythiophene-2, 5-dicarboxanilides (5a–h), obtained by condensation reaction of 3, 4-ethylenedioxythiophene-2, 5-dicarbonyl chloride and corresponding 3- or 4-aminobenzamidines, were evaluated for interactions with double-stranded DNA and RNA, and their cytotoxicity was assayed against the panel of human cancer cell lines. All compounds preferentially bind into the minor groove of DNA and had higher affinity for DNA than for RNA. Compounds 5a–h showed a moderate antiproliferative effect toward the panel of seven carcinoma cells line, whereby the highest inhibitory potential was displayed by compound 5a with unsubstituted amidino moieties in para position.