<p>The oral microbiome has a crucial role in nitric oxide (NO) production and contributes to oral and systemic health. This study compared oral microbiome composition and markers of NO production between highlytrained competitive athletes and inactive controls. Competitive athletes and untrained controls (N = 10 per group) were recruited. Saliva, plasma, supragingival plaque and the tongue dorsum microbiome were sampled. The microbiome was examined using long-read 16S rRNA sequencing and ozone-based chemiluminescence used to measure nitrate (NO₃^-) and nitrite (NO₂^-) levels. Weekly training duration was recorded and aerobic fitness capacity (V̇O_2max) assessed via maximal exercise testing.The beta-diversity of the tongue dorsum microbiome differed between groups (Adonis <i>p</i> = 0.046) and athletes had a higher relative abundance of NO₃^--reducing <i>Rothia mucilaginosa</i> and unclassified <i>Gemella</i> species. No significant differences were detected in the supragingival plaque. Positive correlations were detected between <i>R. mucilaginosa</i> and unclassified <i>Gemella</i> species and aerobic fitness. Athletes had higher levels of salivary NO₃^- (<i>p</i> = 0.003) and NO₂^- (<i>p</i> = 0.03). Exercise training may impact the tongue dorsum microbiome more than supragingival plaque, with the relative abundance of specific health-associated bacteria higher in the tongue dorsum microbiome of athletes. The robust methodologies employed in this study highlight a possible link between consistent exercise and the development of an oral microbiome conducive to health. However, further research is needed to explore the mechanisms connecting exercise, the oral microbiome, and overall health.</p>