BACKGROUND Red ginseng (RG) is processed from Panax ginseng via several methods including heat treatment, mild acid hydrolysis, and microbial conversion to transform the major ginsenosides into minor ginsenosides, which have greater pharmaceutical activities. During the fermentation process using microbial strains in a machine for making red ginseng, a change of composition occurs after heating. Therefore, we confirmed that fermentation had occurred using only microbial strains and evaluated the changes in the ginsenosides and their chemical composition. METHODS To confirm the fermentation by microbial strains, the fermented red ginseng was made with microbial strains (w-FRG) or without microbial strains (n-FRG), and the fermentation process was performed to tertiary fermentation. The changes in the ginsenoside composition of the self-manufactured FRG using the machine were evaluated using HPLC, and the 20 ginsenosides were analyzed. Additionally, we investigated changes of the reducing sugar and polyphenol contents during fermentation process. RESULTS In the fermentation process, ginsenosides Re, Rg1, and Rb1 decreased but ginsenosides Rh1, F2, Rg3, and Compound Y (C.Y) increased in primary FRG more than in the raw ginseng and RG. The content of phenolic compounds was high in FRG and the highest in the tertiary w-FRG. Moreover, the reducing sugar content was approximately three times higher in the tertiary w-FRG than in the other n-FRG. CONCLUSION As the results indicate, we confirmed the changes in the ginsenoside content and the role of microbial strains in the fermentation process.