Background/Objective: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a highly prevalent chronic liver disease with no specific therapeutics. Wuling Powder (WLP) is a classic traditional Chinese medicine prescription with therapeutic potential against MASLD, yet its molecular mechanism remains unclear. This study aims to elucidate the mechanism and possible effective substances of WLP in the treatment of MASLD. Methods: A rat MASLD model was established via high-fat diet feeding to evaluate WLP’s efficacy. Untargeted metabolomics and 16S rRNA sequencing were used to explore the effects of WLP on metabolism and gut microbiota in vivo. Serum pharmacochemistry combined with metabolomics was used to analyze the key active components and core targets of WLP against MASLD, and molecular docking and cell experiments were used to verify the relationship between them. Results: WLP reduced hepatic lipid accumulation and pathological damage, improved lipid levels in blood liver, enhanced antioxidant capacity, and alleviated inflammation in MASLD rats. Mechanistically, WLP regulated 19 metabolic pathways. It also decreased the Firmicutes/Bacteroidota ratio and reduced the abundance of potential pathogenic bacteria (Romboutsia and Turicibacter). Thirty-one WLP-derived components were identified in serum, 13 of which were key active components for treating MASLD. These components, especially 11-deoxyalisol A and 8β-methoxyatractylenolide I, alleviated hepatic steatosis by downregulating NOS2 and PLA2G2A expression. Conclusions: The alleviation of MASLD by WLP was mediated by the regulation of 8 metabolic pathways, alterations in the abundance of Romboutsia and Turicibacter, and the restoration of 20 metabolite levels, an effect primarily ascribed to 13 distinct pharmacodynamic components derived from WLP.