Individual differences in the chronotype, an attitude that best expresses the individual circadian preference in behavioral and biological rhythms, have been associated with cardiometabolic risk and gut dysbiosis. Up to now, there are no studies evaluating the association between chronotypes and circulating TMAO concentrations, a predictor of cardiometabolic risk and a useful marker of gut dysbiosis. In this study population (147 females and 100 males), subjects with the morning chronotype had the lowest BMI and waist circumference (p < 0.001), and a better metabolic profile compared to the other chronotypes. In addition, the morning chronotype had the highest adherence to the Mediterranean diet (p < 0.001) and the lowest circulating TMAO concentrations (p < 0.001). After adjusting for BMI and adherence to the Mediterranean diet, the correlation between circulating TMAO concentrations and chronotype score was still kept (r = −0.627, p < 0.001). Using a linear regression analysis, higher chronotype scores were mostly associated with lower circulating TMAO concentrations (β = −0.479, t = −12.08, and p < 0.001). Using a restricted cubic spline analysis, we found that a chronotype score ≥59 (p < 0.001, R2 = −0.824) demonstrated a more significant inverse linear relationship with circulating TMAO concentrations compared with knots <59 (neither chronotype) and <41 (evening chronotype). The current study reported the first evidence that higher circulating TMAO concentrations were associated with the evening chronotype that, in turn, is usually linked to an unhealthy lifestyle mostly characterized by low adherence to the MD.