Abstract: The ethanol extraction of antibacterial substances from Dictyophora indusial fisscher was optimized using the yield and efficacy of the antimicrobial extract as indicators. On basis of the single factor experimental result, a quadratic general rotary unitized design was applied to finalize the optimization for the process. A regression analysis on the data obtained indicated that the processing temperature exerted the greatest effected on the amount of the extract to be obtained, and it was followed by time and material/solvent ratio. The conditions rendering a maximized yield included a temperature of 72℃, a duration of 1.5 h, and a material/solvent ratio of 1/25. The resulting mushroom extract showed varying antimicrobial activities on Staphylococus aureus, Bacillus thuringiensi, Escherichia coli, Bacillus magaterium, Salmonella pullorum, Salmonella enteritidis, and Bacillus subrilis, with the strongest inhibition effect against S. enteritidis.The research results provide a reference for ntibaterial substances from D.indusial fisscher being developed national food preservative in the industrialization.