Smoking is considered a major risk factor for the development and progression of periodontal diseases. The relation of cigarette smoking to the composition of the subgingival microbiota is not clear. Some studies found higher levels of certain species in smokers, while other studies failed to detect differences in the microbiota between subjects with different smoking histories. The aim of this study is to investigate how smoking in young adults without clinical signs of periodontal disease is associated with the prevalence of subgingival bacteria and periodontal health with the hypothesis that smoking increases the prevalence of the bacteria. Materials and methods 64 periodontally healthy 25 – 35 years old participants were enrolled in this study, 32 in the smokers group and 32 in the non-smokers group. Selection criteria were: age 25 to 35; systemically healthy and periodontally healthy; no periodontal treatment or antimicrobial therapy for at least six months before the examination; at least 20 teeth. Participants filled in a structured questionnaire which recorded their smoking status, general health, socio-economic status and oral health habits. All participants were clinically examined. Four clinical variables were recorded for all participants: aproximal paque index (API), periodontal pocket depth (PPD), bleeding on probing (BoP) and clinical attachment level. The absence of periodontal disease was defined as absence of sites with PPD of >3 mm in any tooth and BoP >0.25. Subgingival plaque samples were collected with sterile paper points from two first molars (16 and 46) for further microbiological analyses. MALDI TOF mass spectrometry was used for bacterial identification. Results 63 different bacterial species from 5 phyla (Actinobacteria, Proteobacteria, Bacteriodetes, Fusobacteria and Firmicutes) were detected in this study. Species from genera Streptococcus, Actinomyces and Veillonela were dominant in this study. Species S. oralis, S. mitis, A. oris, S. sanguinis and V. Parvula showed the greatest prevalence. In average, non-smokers harbored 5 different species and smokers harbored 6 different species. Smokers showed a higher abundance of Fusobacterium phyla compared to non-smokers. No difference in abundance of aerobic bacteria was recorded between smokers and non-smokers, but smokers showed a higher abundance of anaerobic bacteria compared to non-smokers. None of these differences were statistically significant. Smokers showed a higher prevalence of A. odontolyticus (p=0.026) which is acting as a bridging species, enabling co-adhesion between early and late (pathogenic) colonizers. Non-smokers showed a higher prevalence of S. sanguinis (p=0.049) which is beneficial, protective bacteria that showed prominent inhibitory effects on A. actinomycetemcomitans colonization. Conclusion Smoking affects the subgingival microbiota in periodontally healthy young adults and is responsible for the depletion of beneficial bacteria and the increase in pathogenic bacteria. Further studies with a larger sample are needed to enhance our understanding of the exact effect of smoking on the subgingival microbiota.