A novel microbial procedure was proposed for diosgenin production from Dioscorea zingiberensis C.H. Wright (DZW) tubers via employing Aspergillus awamori for the first time. The optimal conditions of fermenter cultivation were established as inoculation dosage of 8%, fermentation temperature of 30 °C, cultivation time of 8 days, initial pH of 7.0 and a stirring rate of 180 rpm when the converted diosgenin content reached a peak value of 74.26 ± 3.23 mg/g substrate. The product was purified by silica gel column and then confirmed as diosgenin (purity: 96.9 ± 2.42%) by nuclear magnetic resonance (NMR). Compared with traditional acid hydrolysis, this new process generated indeed less wastewater with lower chemical oxygen demand (COD) reduced to 500 mg/L from 10,000 mg/L and absence of acid and alkali. This research provided definitely an environmental and high-efficiency microbial technology for diosgenin production.