Cheonggukjang, a popular Korean traditional fermented soyfood, was manufactured by fermenting steamed soybeans in a temperature-controlled room by traditional methods in which steamed soy was exposed to rice straw naturally rich in Bacillus species. B. subtilus and B. licheniformis were found to be the major microorganisms present in cheonggukjang made by the traditional method. We analyzed the composition of 12 kinds of isoflavones and their glycosides present in cheonggukjang collected at various fermentation times. Total isoflavone content in raw soybeans was 2,867 mg/kg and this decreased by about 50% during cooking prior to cheonggukjang preparation. However, total isoflavone content changed slightly during 45 hr of fermentation. Total content of isoflavone glycosides, consisting mainly of daidzin, glycitein, and genistin, decreased by about 40% during 45 hr of fermenting cooked soybeans. The contents of tree isoflavones including daidzein, glycitein, and genistein showed a dramatic increase during fermentation in cheonggukjang preparation, with a 2.9-, 54.0-, and 20.6-fold increase in concentration, respectively, by the end of fermentation (45 hr). In conclusion, short-term fermentation of cooked soybeans with Bacillus species caused conspicuous changes in the composition of isoflavone derivatives, and its implication in terms of health benefits deserves further study.