The intake of oleic acid-rich fats, a hallmark of the Mediterranean diet, has well-documented beneficial effects on human metabolic health. One of the key mechanisms underlying these effects is the regulation of gut microbiota structure and function. However, most existing studies focus on gut bacteria, while gut fungi, as a vital component of the gut microbiota, remain largely unexplored. This study compared the effects of regular peanut oil (PO) and high-oleic acid peanut oil (HOPO) on the gut mycobiome and serum metabolome employing ITS high-throughput sequencing and UPLC-MS/MS metabolomics to explore how dietary fatty acid composition influences gut microecology. Both HOPO and PO effectively reversed high-fat, high-fructose diet (HFFD)-induced reductions in gut fungal diversity, with HOPO showing superior efficacy in restoring gut microbiome balance, as reflected by an improved fungal-to-bacterial diversity ratio and reduced abundance of pathogenic fungi such as