S orghum (Sorghum bicolor) is an important cereal crop as it is generally more stress resistant than other crops, and thus more economical to produce under various environments. Sorghum plants contain various phytochemicals as secondary metabolites or integral cellular components. Our previous study showed that fermented Sorghum extract (FSET) has better antioxidant activity than non-fermented Sorghum extract (SET). Fermentation increased DPPH radical scavenging activities from IC50(μg/Mγ) 481.9 to 28.7 compared with non-fermented condition, with Aspergillus oryzae NK. This study investigated the differences in phytochemical components between SET and FSET caused by fermentation by A. oryzae NK. High-performance liquid chromatography with UV detection and liquid chromatography coupled with electrospray ionization tandem mass spectrometry were used for chemical fingerprinting and the identification of the compounds in SET and FSET. Quercetin-hexose was detected as the major compound in SET, but not in FSET. Fermentation significantly (840%) increased the content of ferulic acid, which was the major compound in FSET. The concentrations of quercetin, apigenin, and luteolin were decreased, whereas the taxifolin content was four times higher in FSET than in SET, and several flavonoids were newly detected after fermentation. In addition, changes in various phytochemicals were detected in FSET compared to those in SET. These results indicate that the increased DPPH radical scavenging activity in FSET is caused by changes in ferulic acid content, which is known as an effective radical scavenger. Thus, fermentation by A. oryzae NK increases DPPH radical scavenging activities in Sorghum extract and greatly affects the chemical composition. This study paves the way for further studies on the various biological activities of FSET and for the application A. oryzae NK-based fermentation systems in health food production.