A new method is described to remove and separate biosurfactants from complex mixtures by compressing and harvesting the liquid surface layer. This method was applied to Bacillus subtilis cultures, in which the lipopeptide antibiotic fengycin as well as the polyketide antibiotic bacillaene were produced. The automated harvesting and collection in a custom-built glass body called 'flounder' was repeated several hundred times. The fengycin concentration in the fractions was found to be four times higher than in the culture centrifugate. Of the overall fengycin, 50% (w/w) were recovered after 300 cycles, 95% (w/w) after 800 harvesting cycles. A separation of fengycin from the less surface-active bacillaene could be achieved due to stronger surface activity of fengycin. The ratio of partition coefficients of fengycin and bacillaene was nine times higher compared to foam fractionation. A stepwise increase of the equilibrium surface tension in the centrifugate from 29 to 33 mN/m indicated a fractionated separation of different surface-active substances. The utilization of cell containing culture broth instead of centrifugate had only slight effects on separation efficiency. These results demonstrate the possibility to separate biosurfactants directly from cultivation without the use of extraction solvents or foam formation.