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Antimicrobials are important adjuncts in the treatment of caries and periodontitis. However, increased bacterial resistance and hypersensitivity reactions to commonly used antimicrobials have led to an increasing demand for safe and natural substances. The objective of this study was to investigate the antibacterial effects of ε-polylysine against oral pathogens <i>Streptococcus mutans</i> and <i>Porphyromonas gingivalis</i>. Broth dilution assay, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analyses were performed to explore the antibacterial effect of ε-polylysine against <i>S. mutans</i> strain ATCC25175 and <i>P. gingivalis</i> strain ATCC332277. For the test solution, ε-polylysine was added to the bacterial suspension to prepare 0.125%, 0.25%, 0.5% and 1% ε-polylysine solutions diluted in broth medium. All four concentrations demonstrated complete inhibition of <i>S. mutans</i> and significantly reduced viable cell counts of <i>P. gingivalis</i> after 24 h. From starting inoculum of 9.15 log CFU/mL, <i>P. gingivalis</i> cell counts reduced to 4.01 log CFU/mL in the 0.125% ε-polylysine treatment group. SEM, CLSM, and the LIVE/DEAD bacterial assay of ε-polylysine application on <i>P. gingivalis</i> biofilm-dentin specimens revealed bacterial cell membrane disruption and irregular cell morphologies. The results indicated satisfactory antibacterial efficacy of ε-polylysine against <i>P. gingivalis</i> and <i>S. mutans</i> in liquid medium and as an application on biofilm-dentin specimens.