Mycosubtilin belongs to the family of lipopeptides. Different isoforms with various antifungal activities can be obtained according to the length and the isomery of the fatty acid. In this work, the activities of the mycosubtilin isoforms were first studied against the pathogen Aspergillus niger, revealing the high activity of the anteiso-C17 isoform. Modification of the mycosubtilin isoform patterns during cultures of the natural strain Bacillus subtilis ATCC 6633 was then investigated through amino acid feeding experiments. In parallel, single-gene knockouts and single-gene overexpression, leading to the overproduction of the anteiso-C15 fatty acid chains, were predicted using informatics tools which provide logical reasoning with formal models of reaction networks. In this way, it was in silico predicted that the single overexpression of the ilvA gene as well as the single knockout of the codY gene may lead to the overproduction of anteiso-C15 fatty acid chains. For the first time, it has been demonstrated that overexpression of ilvA helps to enhance the furniture of odd anteiso fatty acids leading to a favored mycosubtilin anteiso-C17 production pattern (+41%). Alternatively, a knock-out codY mutant led to a higher furniture of even iso fatty acids, leading to a favored mycosubtilin iso-C16 production pattern (+180%). These results showed that increased selective synthesis of particular isoforms of mycosubtilin through metabolic engineering is feasible, disclosing the interest of these approaches for future development of lipopeptide-producing strains.