Surfactin is described as one of the most powerful biosurfactants and is natively produced by Ba-cillus subtilis in notable quantities. In addition to other industrially relevant characteristics, anti-microbial properties were highlighted for surfactin. To investigate this property, stress ap-proaches were carried out with biotechnologically established strains of Corynebacterium glutami-cum, Bacillus subtilis, Escherichia coli and Pseudomonas putida with the highest possible amounts of surfactin. Contrary to the popular opinion, growth-reducing effects were only detectable for B. subtilis and E. coli at the higher gram-per-litre scale, while other antimicrobial biosurfactants, such as rhamnolipids and sophorolipids, showed significantly stronger effects on bacterial growth. As the addition of high amounts of surfactin in defined mineral salt medium reduced the cell growth of B. subtilis by about 40%, the initial stress response at the protein level was ana-lyzed by mass spectrometry, showing an induction of stress proteins under the control of the al-ternative sigma factors σB and σW as well as the activation of LiaRS two-component system. Overall, growth-reducing effects could be demonstrated after addition of high amounts of sur-factin at a high gram-per-litre scale, while absolutely no effects could be detected for the exem-plary bacterial strains C. glutamicum and P. putida, challenging the general claim of the antimi-crobial properties of surfactin.