Omega-3 fatty acids (-3 FAs) are associated with a lower risk of ischemic stroke in patients with atrial fibrillation (AF). Antithrombotic mechanisms may in part explain this observation. Therefore, we examined the association of -3 FAs with D-dimer and beta-thromboglobulin (BTG), markers for activated coagulation and platelets, respectively. The -3 FAs eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA) and alpha-linolenic acid (ALA) were determined via gas chromatography in the whole blood of 2373 patients with AF from the Swiss Atrial Fibrillation cohort study (ClinicalTrials.gov Identifier: NCT02105844). In a cross-sectional analysis, we examined the association of total -3 FAs (EPA + DHA + DPA + ALA) and the association of individual fatty acids with D-dimer in patients with detectable D-dimer values ( = 1096) as well as with BTG ( = 2371) using multiple linear regression models adjusted for confounders. Median D-dimer and BTG levels were 0.340 ug/mL and 448 ng/mL, respectively. Higher total -3 FAs correlated with lower D-dimer levels (coefficient 0.94, 95% confidence interval (Cl) 0.90-0.98, = 0.004) and lower BTG levels (coefficient 0.97, Cl 0.95-0.99, = 0.003). Likewise, the individual -3 FAs EPA, DHA, DPA and ALA showed an inverse association with D-dimer. Higher levels of DHA, DPA and ALA correlated with lower BTG levels, whereas EPA showed a positive association with BTG. In patients with AF, higher levels of -3 FAs were associated with lower levels of D-dimer and BTG, markers for activated coagulation and platelets, respectively. These findings suggest that FAs may exert antithrombotic properties in patients with AF.