The Cheonggukjang used in this study was made with four soybean cultivars grown and harvested at the National Agrobiodiversity Center of the National Academy of Agricultural Sciences: C1 (KLS87248), C2 (Nongrim 51), C3 (GNU- 2007-14613), and C4 (Daewon). The soybeans were inoculated with Bacillus subtilis AFY-2 and fermented for 0, 12, 21, 36, 45, and 60 h in a culture room at 35°C. ABTS and DPPH radical scavenging activities were significantly increased upon fermentation for 12 hours in all samples and maintained or decreased after 21 hour The inhibition of NO production was significantly increased compared to the LPS-treated group, and the C2 sample showed inhibition of NO production at 12 hours of fermentation, and the C3 sample showed inhibition at 21 hours of fermentation. PCA analysis revealed that the ABTS and DPPH radical scavenging activity and NO production inhibitory activity reached peak levels around 12 h fermentation time for all samples. Our results indicate that the optimal fermentation time of the Cheonggukjang sample is 12 hours, confirming the high correlation of all experiments.