Condensation polymerization was used in the preparation of modified guanidine polymers. The polymers were composed of hexamethylene diamine and guanidine hydrochloride with subsequent crosslinking of epichlorohydrin. The two-step synthesis method increased the molecular weight of guanidine polymers, and also enhanced the charge density of the polymer due to the formation of the four-membered rings containing quaternary ammonium groups. This was found to improve antimicrobial activity. The molecular structure of the prepared guanidine polymer was characterized by 2D heteronuclear correlation NMR spectroscopy (C-H COSY). Results indicated that guanidine polymer was a mixture with various structures. UV260 absorption and AFM were applied to reveal the dynamic antimicrobial process of guanidine polymer and morphological change of bacterial cells. Both UV260 absorption and AFM images showed that the antimicrobial mechanism of guanidine polymer was to destruct the cell membrane of bacteria and cause the leakage of intracellular components from bacterial cells. (c) 2008 Elsevier Ltd. All rights reserved.