Reverse micelles extraction of erythromycin and amoxicillin were carried out using the novel Sophorolipids biosurfactant. By replacing commonly used chemical surfactants with biosurfactant, reverse micelle extraction can be further improved in terms of environmental friendliness and sustainability. A central composite experimental design was used to investigate the effects of solution pH, KCl concentration, and sophorolipids concentration on the reverse micelle extraction of antibiotics. The most significant factor identified during the reverse micelle extraction of both antibiotics is the pH of aqueous solutions. Best forward extraction performance for erythromycin was found at feed phase pH of approximately 8.0 with low KCl and sophorolipids concentrations. Optimum recovery of erythromycin was obtained at stripping phase pH around 10.0 and with low KCl concentration. On the other hand, best forward extraction performance for amoxicillin was found at feed phase pH around 3.5 with low KCl concentration and high sophorolipids concentration. Optimum recovery of erythromycin was obtained at stripping phase pH around 6.0 with low KCl concentration. Both erythromycin and amoxicillin were found to be very sensitive toaqueous phase pH and can be easily degraded outside of their stable pH ranges.