Microwave-assisted three-liquid-phase extraction (MATLPE) was applied to extract diosgenin and untransformed saponins in Dioscorea zingiberensis C. H. Wright (DZW) fermentation broth from Trichoderma harzianum. The partitioning behaviours of various steroids were investigated, and the removal of soluble protein, microbial cells and raw herb residuals were studied. A three-liquid-phase system (TLPS) consisting of 30% (w/w) ethanol, 15% (w/w) (NH₄)₂SO₄, 40% (w/w) petroleum ether and water was selected. The optimal microwave extraction parameters were as follows: liquid/solid ratio, 80:1; microwave time, 120 s; and microwave power, 380 W. MATLPE integrated the extraction and separation of diosgenin and the untransformed saponins, the separation of solids and liquids, and the removal of soluble impurities (reducing sugars and proteins) in one step. Compared with other methods, MATLPE consumed smaller amounts of reagents, achieved higher recoveries, and resulted in several-fold higher concentrations of diosgenin and saponin extracts.