This study was conducted to investigate the quality characteristics and changes in isoflavone content of Cheonggukjang with added Chaga mushroom by secondary fermentation at 40 for 48 h with or without a starter, Lactobacillus acidophilus KCTC 3925. Cheonggukjang samples were divided into three groups: Control (unsterilized Cheonggukjang fermented without a starter), NS (unsterilized Cheonggukjang inoculated with L. acidophilus KCTC 3925), and YS (heat-sterilized Cheonggukjang inoculated with L. acidophilus KCTC 3925). The approximate composition of the three types of Cheonggukjang was 49.79-51.44% moisture, 4.54-4.72% crude ash, 43.21-44.37% crude protein, 11.58-13.65% crude fat, 37.41-40.07% carbohydrate, 31.92-33.82% dietary fiber. The mineral content included 5.43– 9.64 mg% Na, 1,792.86–1,824.39 mg% K, 253.69–326.09 mg% Ca, 619.37–691.20 mg% P, 92.59–110.59 mg% Fe, and 0.01–0.02 mg%Cd. Free amino acid contents of NS (2,520.92 mg%) and YS (2,421.94 mg%) were significantly higher than that of the control (2,236.76 mg%). Amino-type nitrogen content for the three types of Cheonggukjang ranged from 837.20-920.27 mg% with no significant difference. Ammonia-type nitrogen content ranged from 137.09-169.36 mg%. Supplement of Cheonggukjang with L. acidophilus KCTC 3925 increased production of aglycone isoflavons compared to that of control. Therefore, our results show that fermenting Chaga Cheonggukjang with L. acidophilus KCTC 3925 leads to improved quality characteristics and increased isoflavone aglycone content.