The present study was performed to evaluate how cellular and humoral immune responses were perturbed by immunization of mixed periodontal bacterial biofilms. Each group of mice was immunizared with 1) Porphyromonas gingivalis (P. gingivalis) grown as a planktonic culture, 2) Fusobacterium nucleatum (F. nucleatum), 3) P. gingivalis grown as a biofilm, or 4) mixed P. gingivalis plus F. nucleatum grown as a biofilm culture, respectively. Immune mouse sera were collected from each mouse. Spleens were harvested to isolate T cells and consequently stimulated with antigen presenting cells and P. gingivalis whole cell antigen to establish P. gingivalis-specific T cell lines. There were no significant differences in the mean anti-P. gingivalis IgG antibody titers among mouse groups. Immunization of mice with pure P. gingivalis biofilm or mixed P. gingivalis plus F. nucleatum biofilm resulted in significant reduction of antibody avidity and opsonophagocytosis function. INF-gammaproduction by P. gingivalis-specific T cell lines was also substantially reduced in mouse groups immunized with the biofilm. It was concluded that P. gingivalis biofilm perturbs the cellular and humoral immune responses in periodontal disease.