Background The subgingival microbiota as well as determination of markers such as associated pathogens is still in the focus of dental research. The aim of this controlled clinical trial was to determine clinical applicability of a newly developed chairside bacterial test (CST) for the most relevant periodontal pathogens. Methods Within 125 participants (100 with periodontitis, 25 healthy) two sulcus fluid samples each were collected and pooled for further analysis. Samples were analyzed with CST and results (positive signals for every pathogen/control) were visually detected by eye. As a reference quantitative polymerase chain reaction (qPCR) was performed. Results The detection limit of CST revealed 1.2 × 104 for Treponema denticola (T.d.) and Tannerella forsythia (T.f.), 2.5 × 104 for Porphyromonas gingivalis (P.g.), 5.3 × 103 for Prevotella intermedia (P.i.), and 5.8 × 104 for Aggregatibacter actinomycetemcomitans (A.a.). Based on this maximum potential of positive detections, the sensitivities of CST in reference to qPCR were: T.d. (91.3%); T.f. (86.3%); P.g. (83.8%); P.i. (85.7%), and A.a. (100%). In regard to the clinical diagnosis, the CST assay and the qPCR method reached a sensitivity of 87.82% and 94%, respectively. The specificity for both methods was 100%. Conclusion This newly developed CST can detect five typical periodontal pathogens with a somewhat lower sensitivity towards qPCR that can be classified as “good.”