Background Exploring longitudinal associations of blood biomarkers with left atrial (LA) structure and function can enhance our understanding of atrial fibrillation (AF) etiopathogenesis. Methods We studied 532 participants of the PREDIMED-Plus trial, a multicenter randomized trial in overweight and obese adults with metabolic syndrome. At baseline, 3 and 5 years after randomization, participants underwent transthoracic echocardiography and provided blood for serum biomarker measurements (propeptide of procollagen type I (PICP), high-sensitivity (hs) troponin T (hsTnT), hs C-reactive protein (hsCRP), 3-nitrotyrosine (3-NT), and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP)). Outcomes of interest included LA peak systolic longitudinal strain (LA PSLS), LA volume index (LAVi), LA function index (LAFi), and LA stiffness index (LASi). We performed cross-sectional and longitudinal analyses to evaluate relationships between log-transformed biomarkers and echocardiographic measurements using multiple linear regression and mixed models. Results The participants in this analysis had a mean age of 65.0 (SD 4.8) years, and 40% were females. At baseline, increased NT-proBNP and hsTnT were associated with larger LAVi and worse LA function as measured by the LAFi, LASi, and LA PSLS. Longitudinally, higher NT-proBNP, but not higher hsTnT, was associated with increased LAVi and worsening LA function. Over 5 years, 1 unit increase in log(NT-proBNP) was associated with steeper decline in LA PSLS (-0.19%, 95% confidence intervals (CI) -0.35%, -0.02%) and greater increase in LAVi (0.28 mL/m, 95% CI 0.10, 0.45) each year. PICP, hsCRP, and 3-NT did not show consistently significant associations with LA outcomes at baseline and through 5 years. Conclusion In an overweight and obese population, higher NT-proBNP was associated with LA volume enlargement and worsening LA function over 5 years. The implications of these findings for the prevention and prediction of AF warrant further investigation.