Background:Porphyromonas gingivalis is found in subgingival biofilm and is associated with periodontal disease. Bacteria in biofilms are able to resist higher antimicrobial concentrations than in suspension. Little is known about the susceptibility of P. gingivalis in biofilms to antimicrobial agents. The effects of chlorhexidine gluconate, minocycline hydrochloride, and metronidazole on P. gingivalis biofilms were examined in vitro.Methods:P. gingivalis strain 381 biofilms were prepared on 32 hydroxyapatite disks. At 0, 24, 72, and 144 hours after perfusion of the three antimicrobial agents, two disks from each device were used to assess the antimicrobial effects by adenosine triphosphate (ATP) bioluminescence, and for morphological investigation by scanning electron microscopy (SEM).Results: Close relationships were found between the results of the ATP analyses and the SEM observations in all groups examined. A significant decrease (P <0.001) in ATP content was found between the chlorhexidine‐treated and control groups. The extracellular matrix structure and P. gingivalis cells were altered in the presence of chlorhexidine. Minocycline hydrochloride also caused decrease (P <0.05) in the ATP content and morphological change on P. gingivalis biofilms. Metronidazole showed no significant efficacy against P. gingivalis biofilms.Conclusion: Chlorhexidine gluconate was effective at reducing the viability of P. gingivalis 381 cells in biofilms, while minocycline hydrochloride and metronidazole exhibited weaker effects. J Periodontol 2003;74:1647‐1651.