Periodontitis is modulated by a complex dysbiotic microbiota, these species stimulate upward the production of pro-inflammatory cytokines such as TNF-α, which, in turn, upregulates the production of bone resorption molecules. Enzymes such as MMP-8 and 9 have been associated with the destructive disease. This study evaluated the composition of periodontal microbiota with the checkerboard hybridization technique and its correlation with TNF-α, MMP-8, and MMP-9 evaluated with ELISA, of 80 patients (45 healthy, and 35 with chronic periodontitis). The frequency of the 18 species evaluated was higher in patients with bone loss compared with control group. TNF-α in gingival crevicular fluid was significantly higher in bone loss group ( p  < 0.01); MMP-8 ( p  = 0.34) by MMP-9 ( p  < 0.05) in bone loss group obtained lower values than in control group. Positive correlation of TNF-α was obtained with Aggregatibacter actinomycetemcomitans (rho = 0.38; p  < 0.01), Fusobacterium nucleatum (rho = 0.25; p  < 0.05) and Porphyromonas gingivalis (rho = 0.26; p  < 0.05); negative correlation of MMP-8 with A. actinomycetemcomitans (rho = 0.26; p  < 0.01), Capnocytophaga sputigena (rho = 0.33; p  < 0.01), and F. nucleatum (rho = 0.21; p  < 0.05); also negative correlation of MMP-9 with F. nucleatum (rho = 0.23; p  < 0.05), P. gingivalis (rho = 0.23; p  < 0.05), and Tannerella forsythia (rho = 0.26; p  < 0.01). TNF-α increased due to the increase in each count of A. actinomycetemcomitans ( β  = 0.57; p  = 0.00). The presence of A. actinomycetemcomitan s ( β  = 1.88; p  = 0.00), Campylobacter rectus ( β  = 0.78; p  = 0.01), F. nucleatum ( β  = 0.65; p  = 0.04), and P. gingivalis ( β  = 0.65; p  = 0.04) significantly increases TNF-α levels. TNF-α in gingival crevicular fluid, despite the minimal amounts collected, is a good biomarker of periodontal disease; since levels of TNF-α increases with the increase of the most harmful species to the periodontium.