Abstract Background Inorganic nitrate, which is abundant in leefy green vegetables and mediates cardioprotective effects through nitric oxide related pathways. The enteral microbiome is an emerging key player in cardiovascular diseases and depends on dietary habits. Whether dietary inorganic nitrate influences atherosclerosis-associated microbiome dependent metabolites like short chain fatty acids (SCFA) and trimethylamine N-oxide (TMAO) has not been elucidated so far. Purpose Aim of this study was to investigate the influence of dietary anorganic nitrate intake on atherosclerosis-associated metabolites and the enteral microbiome composition in a healthy trial cohort. Methods In this double-blind randomized controlled trial, we included 30 healthy volunteers, who either received dietary nitrate (0,12 mmol/kg bodyweight) or placebo (equimolar amounts of sodium nitrate) for 30 days. The microbiome metabolites TMAO and SCFA were analysed. Furthermore, upstream enteral microbiome composition was analysed by 16S-rRNA sequencing at baseline and follow-up. Results Systolic blood pressure significantly decreased after nitrate supplementation (baseline 124,73 mmHg vs follow up 120 mmHg, p < 0.05) with no change in controls. Dietary nitrate supplementation significantly increased TMAO levels (control follow-up 343,88 µg/L vs nitrate follow-up 481,15 µg/L, p < 0.05), while SCFA levels remained unchanged. Mechanistically a change in enteral microbiome composition after dietary nitrate supplementation with decreased abundance of the gram-negative bacteria Akkermansia muciniphila was detected, while overall Shannon diversity, richness and evenness did not differ. Conclusions In conclusion, our results indicate that dietary nitrate supplementation alters the enteral microbiome with impact on proatherogenic metabolites. Further work is warranted to investigate the causal relationship between dietary nitrate, the microbiome, downstream metabolites, and the clinical relevance of those findings in a cardiovascular diseased cohort.