The human gut microbiome and its metabolite Trimethylamine N-oxide (TMAO) are sensitive to the human diet and are involved in the complex pathomechanisms that underpin diabetes, obesity, and cardiovascular diseases. A potential involvement of increased TMAO in atrial fibrillation (AF) manifestation and progression is not clear. We measured TMAO in peripheral blood of 45 AF patients and 20 non-AF individuals (matched for age, sex, BMI, prevalence of hypertension and diabetes). TMAO levels in AF (median [IQR] 3.5 µM [2.51-4.53]) were comparable with those in non-AF individuals (3.62 µM [2.49-5.46]) (p = 0.629). There was no association between TMAO and AF progression phenotypes (p = 0.588). In 35 AF patients, TMAO was additionally measured 12-18 months after AF catheter ablation. TMAO levels at baseline and follow-up were correlated (r = 0.481, p = 0.003), and TMAO was increased independent from the success (restoration of sinus rhythm) of the ablation procedure. The data of this pilot study indicate that TMAO is not generally higher in AF and is not associated with AF progression phenotypes. The observed TMAO increase 12-18 months after AF catheter ablation needs further investigation in a larger cohort.