A series of comb-like ionenes (C-ionenes) with aliphatic side chains were prepared via the Menschutkin reaction from amine, epichlorohydrin, and N, N, N', N'-tetramethyl-1,6-hexanediamine. The structures of the resulting ionenes were characterized by means of charge density, H-1-NMR, and C-13-NMR. The antimicrobial properties of the ionenes were also investigated by measuring the minimum inhibition concentration (MIC) against Escherichia coli (E. coli). The corresponding antimicrobial dynamics were studied by detecting the UV absorbance of the E. coli suspension. It was found that in comparison to the linear ionenes (L-ionenes), C-ionenes exhibited lowerMIC values and faster antimicrobial effect influenced by the side chains. In particular, C-ionene with 4-carbon side chains showed MIC at 7.8 mu g/mL and its aqueous solution (31.2 mu g/mL) inhibited 99.9 % E. coli within 2 min. The morphologies of C-ionenes in aqueous solution were investigated by measuring their particle sizes and zeta potentials. In addition, the anti-mold tests against Aspergillus niger and Chaetomium globosum suggested that the comb-like ionenes were effective at inhibiting the growth of the fungi.