Bacillus subtilis S499 is well-known for its ability to produce two families of surfactant lipopeptides: Iturin A and Surfactin S1. Fermentation optimization for this strain was performed to amplify the surfactant production. Ten active variables were analyzed by two successive Plackett-Burman designs, consisting respectively of 12 and 16 experiments to give an optimized medium. The amount of biosurfactant lipopeptides in the supernatant of a culture carried out in this optimized medium was about five times higher than that obtained in nonoptimized rich medium. The analysis of the surfactant molecules produced in such optimized conditions has revealed the presence of a third family of lipopeptides: the fengycins. The time-dependent production of these three families of molecules in bioreactors showed that surfactin S1 is produced during the exponential phase and iturin A and fengycins during the stationary phase.