The present invention provides a low-cost, efficient method for producing a glycolipid biosurfactant, in particular, lactonic sophorose lipids. This method is characterized by culturing a microorganism capable of producing the biosurfactant under limited oxygen supply. The present invention enables preferential production of lactonic sophorose lipids and facilitates recovery of the lactonic sophorose lipids in a solid form. Further, the present invention enables production of high purity acidic sophorose lipids by hydrolyzing high purity lactonic sophorose lipids produced by the above method. The present invention also provides lactonic sophorose lipids that possess strong antibacterial and antifungal activities, and an antibacterial and/or antifungal agent containing the sophorose lipids.