Azalomycin F (AZF) is a kind of antibiotic with antifungal and antibacterial activities, as well as anti-inflammatory and anti-tumor activities. In this study, we evaluated the effects of AZF on atopic dermatitis (AD) and its possible molecular mechanisms. Mice with 2,4-dinitrofluorobenzene-induced AD-like skin lesions were topically treated with 10-30 mg/kg AZF on their dorsal skin for 12 days. Observations focused on skin lesion scores, the frequency of scratching, and histopathological alterations in the skin. In addition, IgE and inflammatory cytokine levels in serum were assessed. The results indicated that topical application of 10-20 mg/kg AZF could reduce skin lesion scores and scratching frequencies in AD mice, while 15-20 mg/kg AZF decreased epidermal thickness and mast cell infiltration. Additionally, the serum levels of IgE, IFN-γ, IL-4, TSLP and IL-1β were reduced with 10-20 mg/kg AZF treatment. Moreover, RNA-Seq was employed to reveal the potential molecular mechanisms underlying anti-AD effects of AZF. KEGG enrichment analysis revealed that the most significantly differentially expressed genes are predominantly enriched in signaling pathways such as NF-κB and TNF. Protein-protein interaction network analysis identifies the key genes including Il1b, Tnf, and Cxcl1. In summary, 15 mg/kg AZF effectively alleviates the inflammatory response in AD mice, and the potential mechanism may involve the regulation of key signaling pathways like NF-κB and TNF, thereby reducing inflammatory factor levels and eliciting an anti-inflammatory effect. These findings provide valuable scientific evidence for the development of novel natural drugs for the treatment of AD.