The aim of this study was to investigate the synergistic anti-inflammatory and anti-dermatitis effects of grape branch extract (GBE) and Pleurotus eryngii (PEE) combinations on the active immune cells and atopic dermatitis-like skin lesions in mice. The results showed that the combination of GBE (12.5 μg/mL) and PEE (500 μg/mL) led to much stronger inhibitory effects on the production of inflammatory mediators, such as NO and PGE2 than that exhibited by GBE (25 μg/mL) and PEE (1000 μg/mL) alone, even at higher concentrations, in LPS-stimulated RAW 264.7 macrophages. The combination of GBE and PEE synergistically inhibited the production of TNF- and IL-6 in LPS-stimulated RAW 264.7 macrophages and PMA plus A23187-activated HMC-1 cells. Furthermore, combined GBE and PEE had a stronger ameliorative effect than GBE and PEE alone by inhibiting the clinical sores, IgE, and IL-4 on atopic dermatitis-like skin lesions in mice. Collectively, these results suggested that the combination of GBE and PEE produced a synergistic antiinflammatory and anti-atopic dermatitis effect on immune cells and atopic dermatitis-like skin lesions in mice.