Chitosan (CS) and trimethyl chitosan (TMC) solutions, as well as nanoparticles synthesized by ionic gelation:method are studied. Their characterization is focused on determining the charge and antimicrobial properties against common pathogenic microorganisms and Lactobacillus spp., usually found in resident microbiota of vaginal and gastrointestinal tract. Special emphasis is given to the evaluation of antimicrobial activity in relation to the available cationic charge and the presence of nano-sized chitosan in comparison with chitosan macromolecules. In order to investigate the chitosan's antimicrobial mode of action diffusion nuclear magnetic resonance (D-NMR) is used as a novel approach. This technique enables the monitoring of chitosan nanoparticles (CSNP) effects onto healthy Lactobacillus cells. D-NMR results indicate that CSNP interact with the membrane of Lactobacillus cells, causing the perturbation of the membrane wall and lead to the death of the cells, suggesting the mechanism of Lactobacillus inhibition caused by CSNP. Lactobacillus inhibition is also reflected in low minimal inhibitory concentration (MIC). (C) 2015 Elsevier B.V. All rights reserved.