Background Chronic heart failure (CHF) affects approximately 26 million people worldwide. Nearly half of CHF patients develop heart failure with preserved ejection fraction (HFpEF), which is associated with myocardial hypertrophy and fibrosis. Although chronic inflammation was suggested as a critical factor contributing to fibrosis development, a new hypothesis of CHF pathogenesis suggested that altered gut microbiota contributes to leaky gut phenotype development and promotes a systemic inflammatory state. CHF patients have an altered gut microbiome. However, the effect of gut microbiota on fibrosis development in HFpEF patients is not yet known. Thus, this clinical study involving HFpEF patients (n = 47) and healthy volunteers (n = 43) intended to identify the correlations between microbiota changes and fibrosis markers in HFpEF patients. Methods We used 16S rRNA metagenomic sequencing to identify the microbiota changes in HFpEF patients. Myocardial fibrosis was quantified using T1 myocardial mapping by using cardiac magnetic resonance. We also assessed the levels of microbial metabolites—trimethylamin N-oxide (TMAO) and short-chain fatty acids (SCFAs)—and measured bloodstream miRNAs and cytokines. The gut microbiome functions were simulated using PICRUSt algorithm. Results The gut microbial communities of HFpEF patients were markedly different from those of healthy individuals. The abundance of Faecalibacterium, Prevotella, and Pseudomonas was significantly decreased, whereas that of Lachnoclostridium, Blautia, Haemophilus, Dorea, Peptococcus, and Tyzzerella was increased in HFpEF patients. These changes could have affected TMAO metabolism and SCFA production: TMAO and hydroxypyruvate levels were significantly higher, whereas isovaleric, methylbutyric, and propionic acids were significantly lower in HFpEF patients than in healthy individuals. The simulation with PICRUSt revealed that genes responsible for starch fermentation, SCFA production, and secondary bile acid metabolism were downregulated. Correlation analysis identified the involvement of microbiota changes and miRNAs 183-3p and 193b-3p. Conclusions Gut microbiome composition shifts in HFpEF patients impair biochemical functions, increase TMAO production, and decrease SCFA biosynthesis. The significant decrease in Faecalibacterium could have the most prominent effect on the host physiology. However, this needs to be determined by conducting experiments on animal models, because the mechanism by which the microbiota is associated with cardiac fibrosis development is not yet known.