Imperatae Rhizoma belongs to the Poaceae family and is the rhizome of the perennial plant Imperata cylindrica. As a versatile traditional Chinese medicinal herb that is both edible and medicinal, it is used to treat hematuria, damp-heat jaundice, vomiting, and bleeding. Atopic dermatitis (AD) is an inflammatory dermatitis characterized by chronic, recurrent, pruritic conditions. However, the adverse effects of current mainstream therapies include commonly used medications seriously affect the quality of patients’ life. This study investigates the role of Imperatae Rhizoma extract (IRE) in AD and underlying mechanisms. Using a dinitrochlorobenzene-induced AD mouse model, we found that IRE alleviates AD symptoms by reducing skin dryness, itching, and ear swelling, and modulating epidermal thickening and mast cell infiltration. The liquid chromatography-mass spectrometry combined with the self-built database identification revealed 14 components in IRE, primarily consisting of phytosterols and triterpenes. Network pharmacology suggests IRE’s anti-inflammatory effects may involve the phosphatidylinositol 3-kinase/protein kinase B/nuclear factor-κB (PI3K/AKT/NF-κB) pathway. In vitro, IRE mitigated the activation of this pathway, inhibiting NO and inducible nitric oxide synthase (iNOS) expression, and reducing tumor necrosis factor-α and interleukin-6 release. IRE also enhanced human immortalized keratinocyte cell line HaCaT cell migration, promoting skin healing, and showed inhibitory effects on Staphylococcus aureus on the surface of skin. In conclusion, IRE may serve as a potential treatment for AD by reducing inflammation through the PI3K/AKT/NF-κB pathway and regulating skin microbiota. This research not only provides a theoretical basis for the application of IRE in the treatment of AD, but also offers a new direction for the development of food and medicine homologous natural compound-based therapies in dermatology.