This work describes the synthesis and biological activity of novel amidine derivatives of 2-substituted benzazoles, which include benzoxazole, benzothiazole and benzimidazole as heterocyclic nuclei. They were prepared from amidino-substituted precursors: 2-aminophenol, 2-aminothiophenol, bis(2-aminophenyl)-disulfide and 1,2-diaminobenzene. A new method for the synthesis of isomeric amidino-substituted 2-aminophenols starting from cyano-substituted 2-aminophenols by the Pinner reaction was optimized and developed. In the condensation reactions of precursors with carboxylic acids, acyl chlorides and aldehydes, two small libraries of 2-substituted benzazole amidines were prepared for comparison of the effects that the amidine substituent and the heterocyclic nucleus have on biological activity. All compounds were completely spectroscopically characterized, while the molecular structure of some precursors was confirmed by X-ray diffraction. Antiproliferative activity in vitro on four tumor cell lines was determined for the synthesized amidine benzazoles and the interaction of several selected derivatives with polynucleotides using UV-vis, fluorescence and CD spectroscopy was studied. All tested benzoxazoles and benzothiazoles showed strong antiproliferative activity in micromolar inhibition concentrations, which was mostly contributed by the imidazoline amidine substituent, while benzimidazoles were significantly less active.