The species spectrum of the oral microbiome is considered to be the key factor in the development and progression of periodontal inflammatory disorders. The “red complex” including Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola has the highest pathogenic potential. These bacteria have several biochemical mechanisms that allow them to colonize and destroy periodontal tissues. Proteolytic enzymes play a crucial role in this process. Early diagnosis of pathological conditions induced by microbial contamination allows for the timely treatment of periodontitis. Otherwise, the development of the disease may lead to tooth loss. A total of 48 patients aged 18 to 65 years old who required professional oral hygiene were recruited for this clinical study. Microbial content analysis of dental plaque from the interdental space and the back of the tongue was performed using real-time PCR. To determine the proteolytic activity of oral bacteria, the new express diagnostic method was applied (diagnostic sensitivity, 0.875; specificity, 0.928). The results demonstrate a strong and significant correlation between the new method and the PCR analysis (r = 0.785, p < 0.001). These results show that the new express method can be valuable as an early diagnostic method for periodontal inflammatory disorders caused by the “red complex” bacteria.