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Suppression of pathogenic bacterial growth to increase food and agricultural productivity is important. We previously developed novel hexapeptides (KCM12 and KCM21) with antimicrobial activities against various phytopathogenic bacteria and N 2 plasma-treated buffer (NPB) as an alternative method for bacterial inactivation and as an antibiofilm agent of crops. Here, we developed an enhanced antibiofilm method based on antimicrobial hexapeptides with N 2 plasma-treated buffer against plant pathogens. Our results demonstrated that hexapeptides effectively inhibited the growth of Pseudomonas syringae pv. tomato DC3000 ( Pst DC3000) and the biofilm it formed. Potent biofilm formation-inhibiting effects of hexapeptides were observed at concentrations of above 20 µM, and samples treated with hexapeptide above 100 µM reduced the ability of the bacteria to produce biofilm by 80%. 3D confocal laser scanning microscopy imaging data revealed that the antimicrobial activity of hexapeptides was enough to affect the cells embedded inside the biofilm. Finally, combination treatment with NPB and antimicrobial hexapeptides increased the antibiofilm effect compared with the effect of single processing against multilayered plant pathogen biofilms. These findings show that the combination of hexapeptides and NPB can be potentially applied for improving crop production.