The aim of this study was to investigate the subgingival prevalence of six periodontal pathogens in 352 Italian patients with chronic periodontitis. Possible correlations with clinical parameters, age, gender and smoking status were also investigated. At first visit a pooled subgingival plaque sample was obtained for each subject by using the paper-point method. The samples were processed and analysed according to a commercially available quantitative real time polymerase chain reaction assay (Meridol® Perio Diagnostics, GABA International, Switzerland). Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythia (Tf), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn) and Aggregatibacter actinomycetemcomitans (Aa) were investigated. Fn resulted the most frequently detected (95%) while Tf showed the highest load (12x105 cells/plaque sample). Aa was the less represented bacteria for load and presence. Bacterial load of Pg, Td, Tf and Fn showed a direct correlation to Bleeding On Probing (BOP) and presence of suppuration (p=0.0001). The bacterial load was always directly correlated to Probing Pocket Depth (PPD) (p=0.0001). Among the investigated variables, PPD resulted the most important risk indicator for periodontal pathogens. BOP appeared as a risk indicator for Td, Tf, Pg detection. Few studies have described the microbiological pattern of chronic periodontal disease in the Italian population. Considering the different forms of periodontitis, similar investigations in other countries are needed to disclose any microbiological differences among populations, which may lead to more specific approaches to prevention and therapy.