Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) causes bacterial speck of tomato, a widely spread disease that causes significant economical losses worldwide. It is representative of many bacterial plant diseases for which effective controls are still needed. Despite the antimicrobial properties of chitosan has been previously described in phytopathogenic fungi, its action on bacteria is still poorly explored. In this work, we report that the chitosan isolated from shrimp exoskeletons (70 kDa and 78 % deacetylation degree) exerts cell damage on Pto DC3000. Chitosan inhibited Pto DC3000 bacterial growth depending on its concentration, medium-pH, and presence of metal ion (Mg(+2)). Biochemical and cellular changes resulting in cell aggregation and impaired bacterial growth were also viewed. In vivo studies using fluorescent probes showed cell aggregation, increase in membrane permeability, and cell death, suggesting the chitosan antibacterial activity is due to its interaction as a polycation with Pto DC3000 membranes. Transmission electron microscopic analysis revealed that chitosan also caused morphological changes and damage in bacterial surfaces. Also, the disease incidence in tomato inoculated with Pto DC3000 was significantly reduced in chitosan pretreated seedlings, revealing a promising action of chitosan as nontoxic biopesticide in tomato plants. Indeed, a wider comprehensive knowledge of the mechanism of action of chitosan in phytopathogenic bacterial cells will increase the chances of its successful application to the control of spread disease in plants.