The aim of our research was to identify by bacterial genomic DNA analysis the prevalence of five different species of periodontopathogenic bacteria present in the subgingival biofilm, specifically: Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Bacterioides forsytus (Bf), Treponema denticola (Td). For the analysis we used the systematic Multiplex-PCR-microdent kit with species-specific primers. We studied a group of 48 subjects, 18 males and 30 females, from 18 to 78 years of age. The initial clinical screening enabled us to select, among the group analysed, 24 subjects with signs of active periodontopathy (Group A) and 24 patients without identifiable clinical evidence of the disease used as the control group (Group B). Within the two experimental groups (A and B), the test was found to be positive in 75% of subjects from group A, whereas the test was found to be negative in all the subjects from group B. Our research shows that the Multiplex-PCR system is reliable. Furthermore, the sensitivity and simplicity of this technique, as well as the decrease in working times and the possibility of identifying non-culturable bacteria, since the presence of viable organisms is not essential, make this technique indicated for the simultaneous identification of periodontopathogenic bacteria and might, in perspective, provide a more effective clinical alternative to the techniques of bacterial typing of the subgingival plaque.