The present study compared the recovery of six periodontal pathogens by paper point samples from two different aspects of periodontal lesions by quantitative real-time polymerase chain reaction (PCR). Twenty patients with untreated chronic periodontitis were randomized into two groups. Before subgingival instrumentation and after 10 weeks samples in group A were taken first with a paper point half length (HP) of the probing depth, then with a paper point full length (FP) at the same site. In group B sampling sequence was reversed. Analysis by real-time PCR enabled quantification of six bacteria as well as total bacterial count (TBC). Statistical analysis included t test, Kappa, and Spearman's correlations. Higher TBC could be harvested by use of FP than by HP (mean of differences of ln-transformed counts before therapy: -0.791, CI [-1.515, -0.068], SD 0.770, p = 0.034; after therapy: -0.563, CI [-1.151, 0.024], SD 0.625, p = 0.059). The plaque composition regarding total target pathogens was similar for both samples. Both, for TBC as well as for single target bacteria a strong positive correlation was found between HP and FP (Kappa, Spearman correlation: Aggregatibacter actinomycetemcomitans 0.807, 0.778; Fusobacterium nucleatum 0.573, 0.772; Porphyromonas gingivalis 0.733, 0.824; Prevotella intermedia 0.480, 0.756; Treponema denticola 0.807, 0.814; and Tannerella forsythia 0.692, 0.695). The recovery of target pathogens was similar following sampling at various depths of the periodontal lesion.