Biosurfactants are amphipathic molecules that are synthesized by living cells with numerous potential applications in the areas of health and the environment, among others. However, the high production cost limits massive applications of biosurfactants. An interesting approach to reduce its production cost is the replacement of synthetic culture media by agro-industrial residues associated with hydrophobic inducers. Some of these studies and a recent review on the subject reported that biochemically, the productivity of the biosurfactant can be easily increased by adding inducers to the culture medium, which stimulates microbial growth and also triggers the metabolism of biosurfactant production. Biosurfactant inducers are mainly hydrophobic molecules (e.g. olive oil) that are composed of a pool of molecules (e.g. saturated and unsaturated fatty acids, proteins, and vitamins). However, there is little information about the effect of these specific molecules (e.g. oleic acid) on the production and chemical structure of biosurfactants. In this study, the use of hydrophobic inducers (palmitic acid) at different concentrations (1%, 2%, 5%, and 10%) in the production of surfactin by Bacillus subtilis ATTCC 6633 using cassava wastewater as a carbon source was investigated. Production was carried out at 30 °C, 150 rpm for 72 h. In the analysis of the results of all fermentations, the wastewater cassava was able to produce surfactin (37% (w surfactin/w crude surfactin)) when using the inducer palmitic acid (1%, 2%, 5%, and 10%) in addition to of increasing production (30%, 16%, 75% and 86% (w/w)), respectively, still indicated the formation of different surfactin homologues. There was no influence of the addition of inducers on pH and/or biomass. The surfactin produced was able to reduce the surface tension of water from 72.2 to 30-26 mN/m, using all concentrations of inducers respectively. This study confirms that the use of hydrophobic inducers can reflect on different chemical structures, providing a strong basis for exploring new structures and unknown bioactivities.