Objective To screen the best anti-inflammatory active fraction of Yunüjian in vitro, analyze its chemical composition, and explore its substance basis. Methods Yunüjian decoction was extracted by water. The extract was concentrated, separated by D101 macroporous resin and divided into 4 elution fractions:water elution, 30% ethanol-water elution, 60% ethanol-water elution, and 95% ethanol-water elution. The anti-inflammatory activity of Yunüjian water extract and other elution fractions was evaluated by lipopolysaccharide-induced mouse macrophage inflammation model. Cell viability and nitric oxide (NO) production were evaluated by methyl thiazolyl tetrazolium (MTT) and Griess method. Then, we validated the anti-inflammatory activity of the best active fraction screened. Interleukin (IL)-1β, tumor necrosis factor α, and IL-6 levels were detected by enzyme-linked immunosorbent assay (ELISA), cell morphology was observed by dark-field microscopy, and intracellular nuclear factor κ B (NF-κB) p65 was detected by immunofluorescence staining. Finally, the chemical components of the best active fraction were separated by middle chromatogram isolated gel column separation combined with activity tracking method and identified by nuclear magnetic resonance spectrometer. Results The water extract, water elution fraction, 30% ethanol-water elution fraction, and 60% ethanol-water elution fraction of Yunüjian had anti-inflammatory activity. The anti-inflammatory activities of the water elution fraction and 30% ethanol-water elution fraction were weaker than that of the water extract. The anti-inflammatory activity of the 60% ethanol-water elution fraction was the highest, and the 95% ethanol-water elution fraction had no anti-inflammatory activity. The 60% ethanol-water elution fraction was the most effective in inhibiting the changes of cell morphology and the expression and nuclear translocation of NF-κB p65 induced by lipopolysaccharide. Five compounds (sonnerphenolic B, (+)-nyasol, broussonin A, 2,4-dimethylbenzoic acid, and 2,4-dihydroxybenzoic acid) were isolated from the 60% ethanol-water elution fraction, and 3 of them were from Anemarrhena asphodeloides. Conclusion The 60% ethanol-water elution fraction is the best anti-inflammatory active fraction of Yunüjian, and the main anti-inflammatory activity may be owed to Anemarrhena asphodeloides.