Aim: This study aimed to detect and compare the frequency of selected novel periodontal pathogens in chronic periodontitis patients and periodontally healthy subjects using fluorescence in situ hybridization (FISH). Materials and methods: A total of 72 subjects were divided into two groups of 36 patients in each group. The control group comprised subjects with clinically healthy periodontium, and cases are subjects with chronic periodontitis. Clinical parameters like probing pocket depth (PPD), clinical attachment level (CAL), plaque index (PI), and gingival bleeding index (GBI) were recorded, and a subgingival plaque sample was collected using a sterile Gracey curette and was transferred to a vial of transport media containing Tris-ethylenediaminetetraacetic acid buffer (TE buffer) and was sent for FISH analysis. After the procedure, the fluorescently stained bacteria, that is, Filifactor alocis (F. alocis), Selenomonas sputigena (S. sputigena), and Synergistetes were identified and counted from the smear and quantitated using a simple grading. The data was recorded and statistically analyzed. Results: Increased detection frequency of F. alocis, S. sputigena, and Synergistetes in subgingival plaque samples of patients with chronic periodontitis was more significant than in healthy subjects. However, Synergistete's frequency of detection was less than the other two microorganisms. The detection of Synergistetes cluster A was higher than Synergistetes cluster B. A positive correlation was found between F. alocis and clinical parameters like PPD and GBI (p < 0.05). However, a positive correlation could not be established between the clinical parameters and the expression of S. sputigena and Synergistetes. The detection frequency of F. alocis was found to be more preponderant than the other two organisms. Conclusion: The present investigation has deciphered that uncultivated microorganisms like F. alocis, S. sputigena, and Synergistetes are part of microorganisms associated with chronic periodontitis and may play a role in the initiation and progression of periodontal disease. Clinical significance: In addition to conventional nonsurgical and antibiotic therapies, additional therapeutic modalities are being developed in response to recent insights about the origin of the disease. Molecular techniques like FISH will accomplish a quick detection of novel periodontal pathogens, which would enrich patient care by developing.