Three bacterial species pertaining to the red complex (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) and Aggregatibacter actinomycetemcomitans were investigated in relation to the incidence and severity of periodontal disease. A total of 259 patients were included in this study, 179 being diagnosed with periodontal disease. The gingival crevicular fluid samples were obtained from periodontal pockets and the presence and levels of target bacteria were assessed following DNA extraction and real-time quantitative PCR.Our results account for significant positive associations between the number of bacterial species from the red complex coexisting within a patient and several clinical signs (gingival bleeding, inflammation and bone deterioration). A similar positive association was found between bacterial load of the red complex species and the clinical Case Type diagnostic of the periodontal disease, as well as the probing depth with the most evident results for T. denticola. In conclusion, our study, a first for the Romanian population, confirms previous results found elsewhere and finds a possible regional pathogenic specificity for T. denticola as a major factor for periodontitis severity.