Background/Objectives: Whether habitual dietary omega-3 fatty acid intake is reflected in circulating biomarkers of atrial fibrillation (AF)-related pathways is unclear. We assessed whether usual dietary intake of n-3 fatty acids-considered as total, marine-derived, or non-marine-derived-was associated with the trajectories of five serum markers that reflect AF-related mechanistic pathways [N-terminal pro-B-type natriuretic peptide (NT-pro-BNP), high-sensitivity troponin T (hs-TnT), high-sensitivity C-reactive protein (CRP), the C-terminal propeptide of type-I procollagen (PICP), and 3-nitrotyrosine (3-NT)] over 5 years of follow-up. Methods: In 510 participants of the PREDIMED-Plus trial (older Spanish adults with metabolic syndrome), we measured plasma NT-pro-BNP, hs-TnT, CRP, PICP, and 3-NT at baseline and after 3 and 5 years. Energy-adjusted omega-3 intake was assessed with a validated 143-item food-frequency questionnaire. Cross-sectional and 5-year longitudinal associations according to tertiles of omega-3 fatty acid intake were estimated with linear regression and mixed-effects models. Results: Median total omega-3 intake was 2.0 g/day. Total omega-3 intake was not associated with any biomarker, neither cross-sectionally nor longitudinally. Marine omega-3 was directly associated cross-sectionally with 3-NT (highest vs. lowest tertile +28.4%, 95% CI 5.5 to 56.2; p-trend = 0.014) but not longitudinally. Moderate baseline non-marine omega-3 fatty acid intake was associated with a decrease in PICP after 5 years of follow-up. Conclusions: Overall, habitual total omega-3 fatty acid intake was not associated with circulating AF-related pathways. The sporadic association between marine omega-3 fatty acid intake and 3-NT in the cross-sectional assessment and the isolated non-linear association between baseline non-marine omega-3 fatty acid intake and PICP after 5 years warrant further investigation.