Biosurfactant mannosylerythritol lipids (MELs) have great potential in application as an alternative to chemical surfactants in many fields, while the low-efficiency production and new-to-market nature largely limited its real applications. In this study, a high-efficiency fermentation process to furnish MELs with P. aphidis DSM 70725 was developed, involving addition of iron salts and Tween 20. Concentration, productivity and conversion yield of the MELs was improved to 61 g L-1, 0.255 g L-1 h-1 and 0.68 g g-1, respectively, which have about 100% improvement over the regular fermentation process. This newly developed strategy was then used to fermentatively produce MELs at the pilot-scale (1, 000 L). In addition, MELs were found to possess excellent emulsification activity against xylene, and have phenomenal antimicrobial activity against U. maydis and C. albicans which is not influenced by pH, salinity or initial cell density. Accordingly MELs appear to be superior alternatives to the chemical surfactants traditionally employed in the pesticide industry.