Oral treponemes are related to chronic periodontitis, but the effect of periodontal therapy on the majority of treponemal species is unknown. The aim of this prospective study was to evaluate the dynamics in prevalence profiles of treponemes in different habitats of the oral cavity. Thirty-five patients with chronic periodontitis were randomly assigned to mechanical debridement alone (control group) or systemic amoxicillin/metronidazole plus chlorhexidine (test group). Subgingival and mucous membrane plaque samples were taken at baseline, after 10 days, and during supportive periodontal therapy at 3, 6, 9, 12, 18, and 24 months. T. denticola, T. lecithinolyticum, T. maltophilum, T. socranskii, T. vincentii, and treponemal phylotypes I–VII were detected using polymerase chain reaction (PCR) and dot blot analysis. For the majority of the assessed treponemes, a significant intragroup increase in prevalence in the different habitats (P<0.05) occurred over the study course but, compared to debridement alone, adjunctive antimicrobial therapy resulted in a nonsignificant trend toward lower prevalence in the subgingival habitat. In no case were treponemes eradicated from the oral cavity. After both therapies, possibly new infection with and/or dissemination of Treponema ssp. occurred, which led to treponemes recovering in different habitats and to increased intraoral prevalence. The prescribed adjunctive antimicrobial therapy may limit this increase in the subgingival region.