This study was performed to investigate the antimicrobial effect of the Pulsatilla koreana extracts against food-borne pathogens. First, the Pulsatilla koreana was extracted with methanol at room temperatures, and fractionation of the methanol extracts from Pulsatilla koreana was carried out by using petroleum ether, chloroform, and ethyl acetate, and methanol respectively. The antimicrobial activity of the Pulsatilla koreana extracts was determined using a paper disc method against food-borne pathogens and food spoilage bacteria. The ethyl acetate extracts of Pulsatilla koreana showed the highest antimicrobial activity against Staphylococcus aureus, Salmonella enteritidis and Shigella dysenteriae. The Staphylococcus aureus and Shigella dysenteriae were inhibited by petroleum ether and chloroform extracts of Pulsatilla koreana as well as ethyl acetate extracts of Pulsatilla koreana. The synergistic effect has been found in combined extracts of Pulsatilla koreana and Portulaca oleracea as compared to each extracts alone. Finally, the growth inhibition curve was determined using ethyl acetate extracts of Pulsatilla koreana against Staphylococcus aureus and Shigella dysenteriae. The ethyl acetate extract of Pulsatilla koreana showed strong antimicrobial activity against Staphylococcus aureus at the concentration of 2,000 ppm. The 2,000 ppm of ethyl acetate extract from Pulsatilla koreana retarded the growth of S. aureus more than 12 hours and Shigella dysenteriae up to 9 hours.