Abstract Background Left ventricular diastolic dysfunction (LVDD) is an important precursor of heart failure (HF), but little is known about its relationship with gut dysbiosis and microbial-related metabolites. By leveraging the multi-omics data from the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), a study with population at high burden of LVDD, we aimed to characterize gut microbiota associated with LVDD and identify metabolite signatures of gut dysbiosis and incident LVDD. Results We included up to 1996 Hispanic/Latino adults (mean age: 59.4 years; 67.1% female) with comprehensive echocardiography assessments, gut microbiome, and blood metabolome data. LVDD was defined through a composite criterion involving tissue Doppler assessment and left atrial volume index measurements. Among 1996 participants, 916 (45.9%) had prevalent LVDD, and 212 out of 594 participants without LVDD at baseline developed incident LVDD over a median 4.3 years of follow-up. Using multivariable-adjusted analysis of compositions of microbiomes (ANCOM-II) method, we identified 7 out of 512 dominant gut bacterial species (prevalence > 20%) associated with prevalent LVDD (FDR-q < 0.1), with inverse associations being found for Intestinimonas_massiliensis, Clostridium_phoceensis, and Bacteroide_coprocola and positive associations for Gardnerella_vaginali, Acidaminococcus_fermentans, Pseudomonas_aeruginosa, and Necropsobacter_massiliensis. Using multivariable adjusted linear regression, 220 out of 669 circulating metabolites with detection rate > 75% were associated with the identified LVDD-related bacterial species (FDR-q < 0.1), with the majority being linked to Intestinimonas_massiliensis, Clostridium_phoceensis, and Acidaminococcus_fermentans. Furthermore, 46 of these bacteria-associated metabolites, mostly glycerophospholipids, secondary bile acids, and amino acids, were associated with prevalent LVDD (FDR-q < 0.1), 21 of which were associated with incident LVDD (relative risk ranging from 0.81 [p = 0.001, for guanidinoacetate] to 1.25 [p = 9 × 10⁻⁵, for 1-stearoyl-2-arachidonoyl-GPE (18:0/20:4)]). The inclusion of these 21 bacterial-related metabolites significantly improved the prediction of incident LVDD compared with a traditional risk factor model (the area under the receiver operating characteristic curve [AUC] = 0.73 vs 0.70, p = 0.001). Metabolite-based proxy association analyses revealed the inverse associations of Intestinimonas_massilliensis and Clostridium_phoceensis and the positive association of Acidaminococcus_fermentans with incident LVDD. Conclusion In this study of US Hispanics/Latinos, we identified multiple gut bacteria and related metabolites linked to LVDD, suggesting their potential roles in this preclinical HF entity.