Nowadays, rising environmental awareness and need for replacement of petroleum derived chemicals drive the production processes towards more bio-based and green economy. Microbial production of detergents is one excellent example of this rising trend. Among biosurfactants (BSs) produced by various yeast and bacteria, glycolipids are the most promising ones due to their attractive properties and high product titers. Behind these high titers are both dedicated natural biosynthetic processes and tailored metabolic pathways that drive the well-oiled machinery of the most potent producers. Besides providing some structural information, this chapter unravels the genetic organization and subsequent regulatory mechanism of the microorganisms that are deployed to produce rhamnolipids (RLs), trehalose lipids (TLs), sophorolipids (SLs), mannosylerythritol lipids (MELs) and cellobioselipids (CBLs). As many of these glycolipids can be considered as secondary metabolites, the regulatory aspects of their synthesis are briefly discussed. Furthermore, the various ways in which genetic modification has pushed the boundaries of each microorganism are discussed. Conclusively, some future perspective points out the advancements in the field of genetic engineering that will overcome the limitations and contribute to the further establishment of glycolipid applications in various industrial sectors.