We assessed the level of evidence for the presence of new periodontal pathogens by (i) comparing the occurrence of non-classical periodontal taxa between healthy vs. periodontitis patients (Association study); (ii) assessing the modifications in the prevalence and levels of these species after treatments (Elimination study). In the Association study, we compared the prevalence and levels of 39 novel bacterial species between periodontally healthy and periodontitis patients. In the Elimination study, we analyzed samples from periodontitis patients assigned to receive scaling and root planing alone or with metronidazole+ amoxicillin TID/ 14 days. Levels of 79 bacterial species (39 novel and 40 classic) were assessed at baseline, 3 and 12 months post-therapy. All samples were analyzed using Checkerboard DNA-DNA hybridization. Out of the 39 novel species evaluated, eight were categorized as having strong and four as having moderate association with periodontitis. Our findings suggest strong evidence supporting Lancefieldella rimae, Cronobacter sakazakii, Pluralibacter gergoviae, Enterococcus faecalis, Eubacterium limosum, Filifactor alocis, Haemophilus influenzae, and Staphylococcus warneri, and moderate evidence supporting Escherichia coli, Fusobacterium necrophorum, Spiroplasma ixodetis, and Staphylococcus aureus as periodontal pathogens. These findings contribute to a better understanding of the etiology of periodontitis and may guide future diagnostic and interventional studies.