This study investigated the impact of tobacco exposure, specifically through heating and burning, on periodontopathogens in the subgingival microbiome among clinically healthy individuals and those diagnosed with periodontitis. The sample comprised 66 subjects (26–56 years, median 38 yrs; 64% females) classified as non-smokers, classic cigarette smokers, and tobacco heating system (THS) smokers (each N = 22). Full-mouth periodontal examination was performed, and 330 paper-point samples from periodontal pockets were collected. Next-generation sequencing of 16S rRNA genes was conducted to identify the composition of subgingival microbiome. Periodontitis prevalence among the groups was ranked as THS (41%) < non-smokers (44%) < cigarette smokers (68%), without statistically significant differences between the groups. The number of perio-pathogenic species was higher in subjects with periodontitis compared to those without (median 7 vs. 6 species; p = 0.005) but without significant differences between exposure groups: non-smokers (6) = smokers (6) < THS (6.5). When combining exposure and periodontal status, each smoker group had more perio-pathogenic species than non-smokers: non-smokers without periodontitis (5) < smokers without periodontitis (5.5) < THS without periodontitis (6); non-smokers with periodontitis (6.5) < THS with periodontitis (7) = smokers with periodontitis (7). Multiple linear regression indicated periodontitis as the sole predictor of perio-pathogenic species quantity, irrespective of the type of tobacco consumption, sex, age, or oral hygiene (R2 = 0.163; p = 0.005). In conclusion, the quantity of perio-pathogenic species in the subgingival microbiome was more influenced by periodontitis than by exposure to tobacco smoke, regardless of whether it was heated or burned.