The diagnosis of periodontal disease is mainly based on use of clinical and radiographic evidence. In this study we employed a quantitative PCR analysis of Aggregatibacter actinomycetemcomitans and Treponema denticola as species strongly involved in periodontal diseases, burden in periodontal pockets to detect the main sampling factors that interfere with qPCR results. From 22 patients with advanced periodontal disease, subgingival plaque was comparatively collected by paper points and periodontal Gracey curettes. Samples were collected from the same situs in presence of gingival bleeding and absence of bleeding. The concordance and agreement of results between samples were assessed. The present study demonstrates that subgingival plaque sampling with sterile absorbable paper points is often accompanied by gingival bleeding resulting in quantification biases of periodontal pathogens.