The prebiotic properties of human milk oligosaccharides (HMOs) and emerging evidence of immunomodulatory effects suggest their potential therapeutic value in allergy management. 2'-Fucosyllactose (2'-FL) has been reported to alleviate food allergies, while the effect of other fucosylated HMOs on food allergy remains unclear. In this study, we assess the effect of two HMOs, 2'-FL and 3-fucosyllactose (3-FL), on symptomatology and immunological responses in an ovalbumin (OVA)-sensitized mouse model of food allergy as well as their influence on gut microbiota. The assessment of allergic symptoms, specific immunoglobulin E (IgE), and related gene expression levels in sensitized mice indicated that 3-FL was as effective as 2'-FL in alleviating food allergy. 2'-FL and 3-FL significantly decreased serum levels of OVA-specific IgE, mouse mast cell protease (mMCP-1) and IL-4 while increasing the levels of IFN-γ. Additionally, 2'-FL and 3-FL down-regulated gene expression of allergy-related cytokines in the small intestine and improved intestinal barrier damage. Furthermore, both 2'-FL and 3-FL treatment positively influenced the gut microbial profiles, in particular by enhancing the proportion of beneficial bacteria such as