Background: The collection of subgingival plaque samples with paper points is time-consuming and accident-sensitive. However, the collection of saliva is simple and contains pathogens of all intraoral surfaces. The aim of this study is to investigate whether a sampling strategy with mouthrinse (mouthrinse sample [MSP]; test) leads to results comparable with standard sampling method (multiple site test from the deepest pocket of each quadrant [MT4]; control). Methods: In 50 patients with periodontitis, subgingival plaque was sampled from the deepest pocket of each quadrant by using paper points and by gaining saliva with saline mouthrinse. Analysis was performed using a commercially available polymerase chain reaction test for 11 periodontal pathogens. Results: Detection frequency of Aggregatibacter actinomycetemcomitans (MT4/MSP: 42%/36%), Porphyromonas gingivalis (78%/66%), Tannerella forsythia (98%/84%), Treponema denticola (94%/74%), Parvimonas micra (86%/62%), Campylobacter rectus (90%/76%), Eubacterium nodatum (64%/30%), Prevotella intermedia (58%/54%), and Eikenella corrodens (90%/82%) was higher with MT4 than MSP. For Fusobacterium nucleatum (100%/100%), there was no difference between test and control. Only detection frequency of Capnocytophaga species (68%/74%) was higher with MSP than MT4. Differences were significant for P. gingivalis, T. forsythia, T. denticola, P. micra, C. rectus, and E. nodatum. Conclusions: There is no significant difference between MT4 and MSP for detection frequency of key pathogen A. actinomycetemcomitans. Key pathogens P. gingivalis, T. forsythia, T. denticola, P. micra, C. rectus, and E. nodatum show statistically higher detection frequencies with MT4.