ObjectivesCulturable and unculturable microorganisms have been associated with periodontitis. Their differential proportions and composition have not been evaluated by their severity and complexity defined by stages in the 2018 AAP-EEP classification.MethodsOne hundred eighty subgingival biofilm samples were collected in Spain and Colombia from subjects categorized as health/gingivitis: periodontitis stages I/II periodontitis stages III/IV. Target culturable microorganisms (Porphyromonas gingivalis,Aggregatibacter actinomycetemcomitans, Tannerella forsythia,Treponema denticola, andEubacterium nodatum)and target unculturable microorganisms (Filifactor alocis,Eubacterium saphenum,Eubacterium brachy,Desulfobulbus oralis)were evaluated by quantitative PCR analysis. In addition, their differences and association with periodontal status were analyzed by ANCOVA and logistic regression models once adjusted to age, current smoking, and country.ResultsP. gingivaliswas significantly associated with periodontitis stages I/II, OR 2.44 (CI 95% 1.08–5.47) and stages III/V, OR 6.43 (CI 95% 2.43–16.9).T forsythia, OR 7.53 (CI 95% 2.07–27.4);D. oralis, OR 5.99 (CI 95% 2.71–13.23);F. alocis, OR 10.9 (CI 95% 4.56–23.2);E. brachy, 3.57 (CI 95% 1.40–9.11); andE. saphenum, 4.85 (CI 95% 1.99–11.7) were significantly associated only with stages III/IV periodontitis.P. gingivalisevidenced significant differences with the increase in the severity of the periodontal lesion: 2.97 colony forming unit (CFU)/μL (CI 95% 2.32–3.54) health/gingivitis, and 4.66 CFU/μL (CI 95% 4.03–5.30) and 5.90 CFU/μL (CI 95% 5.20–6.48) in stages I/II and III/IV respectively (p< 0.0001). Unculturable microorganisms only evidenced differences in concentration in stages III/IV compared with health-gingivitis (p≤ 0.001).ConclusionCulturable and unculturable are strongly associated with stages III/IV periodontitis. Classic culturable microorganisms are more sensitive to differentiate between stages of periodontitis in the quantitative analysis.Clinical relevanceFuture interventional studies of periodontal disease should includeFilifactor alocis,Eubacterium saphenum,Eubacterium brachy, andDesulfobulbus oralisas possible markers of therapy response and as indicators of progressive disease.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00784-023-04907-5.