Polyhexamethylene biguanide (PHMB) is a cationic biocide. The antibacterial mode of action of PHMB (at concentrations not exceeding its minimal inhibitory concentration) upon Listeria innocua LRGIA 01 was investigated by Fourier transformed infrared spectroscopy and fluorescence anisotropy analysis. Fourier transformed infrared spectra of bacteria treated with or without PHMB presented some differences in the lipids region: the CH(2)/CH(3) (2924 cm(-1)/2960 cm(-1)) band areas ratio significantly increased in the presence of PHMB. Since this ratio generally reflects membrane phospholipids and membrane microenvironment of the cells, these results suggest that PHMB molecules interact with membrane phospholipids and, thus, affect membrane fluidity and conformation. To assess the hypothesis of PHMB interaction with L. innocua membrane phospholipids and to clarify the PHMB mode of action, we performed fluorescence anisotropy experiments. Two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and its derivative 1-[4-(trimethyl-amino)-phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), were used. DPH and TMA-DPH incorporate inside and at the surface of the cytoplasmic membrane, respectively. When PHMB was added, an increase of TMA-DPH fluorescence anisotropy was observed, but no changes of DPH fluorescence anisotropy occurred. These results are consistent with the hypothesis that PHMB molecules perturb L. innocua LRGIA 01 cytoplasmic membrane by interacting with the first layer of the membrane lipid bilayer.