This study aimed to explore the potential mechanisms of farnesol in the protection of the intestinal epithelium barrier from invasion by Candida albicans (C. albicans) via untargeted metabolomics. The C. albicans reference strain SC5314 and Caco-2 cells were used in this study. The effect of different concentrations of farnesol on the co-culture of C. albicans and Caco-2 cells was investigated using the CCK-8 assay. The effect of farnesol on C. albicans biofilm formation was also observed. There were 4 treatment groups, including the Caco-2 + C. albicans (group 1), Caco-2 (group 2), Caco-2 + C. albicans + farnesol (group 3), and a quality control (QC group) for metabolite extraction, followed by LC-MS/MS analysis and bioinformatics analysis. Farnesol treatment significantly reduced the adhesion of C. albicans and inhibited the formation of C. albicans biofilm. A total of 22 differential metabolites were identified in group 1 vs. group 2, such as acetylcarnitine, linoleic acid, spermidine, and glutathione disulfide. These differential metabolites were involved in fatty acid biosynthesis, linoleic acid metabolism, biosynthesis of unsaturated fatty acids, and glutathione metabolism. There were 18 differential metabolites identified in group 3 vs. group 1, including acetylcarnitine, hypoxanthine, L-glutamate, and linoleic acid, which were enriched in fatty acid biosynthesis, linoleic acid metabolism, and biosynthesis of unsaturated fatty acids. C. albicans can damage the intestinal barrier by affecting the metabolism of acetylcarnitine, linoleic acid, glutathione. Farnesol may protect the intestinal epithelium barrier from the invasion of C. albicans by regulating the metabolism of acetylcarnitine, linoleic acid, and L-glutamate.