Mastic is a natural resin collected from the Mastic tree, which exists only on the Greek island of Chios. Our previous study has shown that oral care with mastic gel in dogs significantly reduces the activity of periodontal disease bacteria, however, scientific evidence of mastic efficacy still has to be examined. The aim of this study is to identify the novel efficacy of Mastic by focusing on the anti-allergic effect in a mouse model of atopic dermatitis (AD). A mouse mode of AD was generated in female NC/nga mice by repetitive topical sensitization with toluene diisocyanate (TDI). Topical treatment of 3% or 5% Mastic gel daily onto ear auricle and back skin during the experiment. Thickness of back skin, AD score, trans epidermal water loss (TEWL), and itch behavior were measured weekly. One day after the last TDI exposure, all mice were euthanized and serum, auricular lymph node and skin tissue were sampled for histology, cytokine determination and FACS analysis. Mastic treatment significantly attenuated thickness of skin, AD score, TEWL and itch behavior. Corroborated reduction was seen in the numbers of T cells and IgE-B cells as well as pro-inflammatory cytokine production. Our findings demonstrated that topical Mastic treatment significantly ameliorates the inflammatory and pruritic responses in a mouse model of AD.