Antimicrobial activity of sophorolipid produced by Candida bombicola ATCC 22214 was investigated, Minimum inhibitory concentration of sophorolipid to Propionibacterium acne and Bacillus subtilis were 0.5 ppm and 4 ppm, respectively. However no antimicrobial activity to E. coli was detected. Indicating the antimicrobial activity of sophorolipid only toward gram positive bacteria. Antifungal activity of sophorolipid was also detected in the hyphae growth assay for Botrytis cinerea. Malate dehydrogenase, a cytoplasmic enzyme, of B. subtilis was remarkably increased with the sophorolipid treatment, indicating the leakage of membrane by sophorolipid. This result shows the potentials of sophorolipid as mild, non-toxic antimicrobial agent.