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Periodontal disease is considered to be a biofilm infectious disease. The effects of macrolide and tetracycline on biofilm were examined in in vitro biofilm model made of periodontal disease-associated bacteria. Biofilms were made on salivary pellicle by adding Streptococcus gordonii for 2 days, followed by Porphyromonas gingivalis inoculation for 2, 5, or 12 days. Biofilms were treated with macrolide antibiotics; erythromycin (EM), azithromycin (AZM) and josamycin (JOM) and tetracycline antibiotic, minocycline (MINO). The effects of these antibiotics on biofilms were examined using colorimetric quantification method, scanning electron microscope (SEM) and confocal laser scanning microscopy (CLSM). When antibiotics were added to the biofilm 2 days after inoculation of Porphyromonas gingivalis (biofilm inhibition model), all four antibiotics decreased the number of bacteria by both colorimetric method and SEM observation. When antibiotics were added to biofilms 5 or 12 days after inoculation (biofilm destruction model), those in biofilms were decreased by EM and AZM compared with JOM and MINO. Moreover, CLSM observation demonstrated that EM and AZM killed bacteria in biofilm more deeply than JOM and MINO. These results suggest the feasibility of EM and AZM for the treatment of periodontal disease as a biofilm infectious disease.