Microbial biosurfactants are surface active metabolites synthesized by microbes growing on a variety of substrates. In spite of having great potential for commercial, therapeutic and environmental applications, industrial level production has not been realized for their low yields and productivities. One vital factor determining their biosynthesis is the genetic makeup of the producer organisms. Studies on molecular genetics and biochemistry of the synthesis of several biosurfactants have revealed the operons, the enzymes and the metabolic pathways required for their extracellular production. Surfactin, a cyclic lipopeptide biosurfactant is a potent antimicrobial agent and is produced as a result of non-ribosomal biosynthesis catalyzed by a large multienzyme peptide synthetase complex called the surfactin synthetase. Pathways for the synthesis of other lipopeptides such as iturin, lichenysin and arthrofactin are also mediated by similar enzyme complexes. These non-ribosomal peptide synthetases (NRPSs) responsible for lipopeptide biosynthesis display a high degree of structural similarity among themselves even from distant microbial species. Plasmid-encoded- rhlA, B, R and I genes of rhl quorum sensing system are required for production of glycolipid biosurfactants by Pseudomonas species. Molecular genetics of biosynthesis of alasan and emulsan by Acinetobacter species and of the fungal biosurfactants such as mannosylerythritol lipids (MEL) and hydrophobins have been deciphered. However, limited genetic information is available about biosynthesis of other biosurfactants such as viscosin, amphisin and putisolvin produced by some strains of Pseudomonas species. Understanding of the genetic regulatory mechanisms would help to develop metabolically engineered hyper-producing strains with better product characteristics and acquired capability of utilizing cheap agro-industrial wastes as substrates. This article thus provides an overview of the role and importance of molecular genetics and gene regulation mechanisms behind the biosynthesis of various microbial surfactants of commercial importance.