The probiotic yeast Saccharomyces boulardii has demonstrated potential in obesity intervention, both as a standalone therapy and as a complementary strategy to exercise. Recent studies suggest that S. boulardii may enhance exercise-induced weight loss by improving metabolic efficiency and gut barrier function. Notably, clinical trials have shown that S. boulardii supplementation combined with aerobic exercise can significantly improve body composition parameters beyond exercise alone, likely through its modulation of exercise-induced gut microbiota alterations. However, its efficacy is often limited by low survival rates under gastrointestinal (GI) stress. In this study, we investigated whether exogenous fatty acid supplementation could augment S. boulardii’s GI tolerance and therapeutic effects. Pre-culturing S. boulardii with linoleic acid (LA) significantly increased its GI stress resistance from 47.1% to 60.4%. In obese mice, LA-pre-treated S. boulardii administration resulted in greater weight reduction compared to untreated yeast, mimicking the synergistic effects observed in exercise-probiotic combined therapies. Fecal microbiome analysis revealed that LA-pre-treated S. boulardii elevated the relative abundance of fungal yeast populations and beneficial bacterial genera (e.g., Lactobacillus, Bifidobacterium), suggesting improved probiotic survival and gut microbiota modulation. These findings demonstrate that fatty acid pre-treatment enhances S. boulardii’s anti-obesity effects by boosting its GI survivability and enriching probiotic microbiota, offering a novel strategy to optimize probiotic-based therapies for metabolic disorders, including those adjunct to exercise regimens.