Periodontal disease involves complex interactions of microorganisms and host defenses. This work investigated the associations between putative bacterial pathogens, herpesviruses and chronic periodontitis. Subgingival samples were collected from 40 periodontally healthy individuals and from 40 patients with chronic periodontitis with probing depths of < or =3 mm or > or =6 mm. Multiplex and nested polymerase chain reactions were used to identify bacterial pathogens and herpesviruses. Porphyromonas gingivalis, Tannerella forsythia, Epstein-Barr virus (EBV) type 1, cytomegalovirus (CMV), Aggregatibacter actinomycetemcomitans and EBV type 2 were detected in, respectively, 95, 75, 72.5, 50, 12.5 and 10% of sites with probing depths > or =6 mm. P. gingivalis, T. forsythia, EBV-1 and CMV were statistically associated with probing depths > or =6 mm. A. actinomycetemcomitans and EBV-2 showed no association with periodontitis sites, and no significant associations were found for any of the test infectious agents and probing depths < or =3 mm. Our results confirm an association between P. gingivalis, T. forsythia, EBV-1 and CMV, and chronic periodontitis. These infectious agents may play an important synergistic role in the pathogenesis of chronic periodontitis.