Rhamnolipids are biosurfactants produced by the soil bacterium Pseudomonas aeruginosa. In addition to their high industrial potential as surface-active molecules, rhamnolipids also have antimicrobial properties. In densely populated habitats, such as the soil, production of antimicrobial compounds is important to inhibit growth of competitors. For the latter, it is crucial for survival to sense and respond to the presence of those antibiotics. To gain a first insight into the biological competition involving biosurfactants, we investigated the cellular response of the model organism Bacillus subtilis upon exposure to rhamnolipids by genome-wide transcriptional profiling. Most of the differentially expressed genes can be assigned to two different regulatory networks: the cell envelope stress response mediated by the two-component system LiaRS and the extracytoplasmic function σ factor σM and the CssRS-dependent secretion stress response. Subsequent phenotypic analysis demonstrated a protective function of LiaRS and σM against cell lysis caused by rhamnolipids. Taken together, we present the first evidence that a single antimicrobial compound can simultaneously induce genes from two independent stress stimulons.