Gwakhyangjeonggisan (GS) is composed of 11 kinds of medicinal herbs that may affect the human gut microbiome. GS has been used in traditional Korean medicine for the treatment of irritable bowel syndrome with diarrhea. 20 healthy adult volunteers which is composed of 17 males and 3 females were recruited for the experiment. Volunteers took the GS daily for 2 weeks and collected stool samples before and after. Bacterial 16S rRNA gene was amplified from stool metagenomics DNA and sequenced using a next generation sequencing (NGS) machine to analyze the gut microbiota. NGS data were analyzed by bioinformatics programs such as QIIME, PICRUSt, Cytoscape, and Calypso. 2 subjects (10%) complained of diarrhea after taking the traditional medicine, although the unique phylogenetic position of microbiota that previously existed did not change significantly after taking GS. Gut microbiota compositions of these subjects originally showed less than 55% of genera Prevotella and Bacteroides, which are known to be involved in digestion of food in human gut. “Glycan biosynthesis/metabolism” and “Replication/repair” functions in the gut microbiota of the diarrhea subjects tended to decrease compared to asymptomatic subjects. On the other hand, “Cell motility” and “Membrane transport” tended to increase. In conclusion, GS can negatively affect the human gut microbiota in subjects which have less than 55% of genera Prevotella and Bacteroides. It is necessary for accumulating database to understand whether the traditional Korean herbs should be prescribed separately according to the human gut microbiota.