Genistein, a soy isoflavone, has been widely used as an anti-inflammatory agent in vitro and in vivo. Although it exhibits antibacterial activity against several bacterial species, its effect on periodontal bacteria remains unknown. This study aimed to investigate the antibacterial activity of genistein against two typical periodontal pathogens, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. The broth microdilution method revealed that genistein inhibited bacterial growth at minimum inhibitory concentrations of 80?160 μM. Viable bacterial counts further confirmed its bactericidal activity against P. gingivalis and A. actinomycetemcomitans, as evidenced by decreased bacterial counts in the presence of genistein. Remarkably, the viability of YD15, a human oral epithelial cell line, was only slightly reduced at a high concentration of genistein. These findings indicate that genistein demonstrates antibacterial activity against periodontal pathogens, which could bear significant clinical implications for periodontal treatment.