AbstractUltrafiltration was used in a one‐step method to purify and concentrate biosurfactants—surfactin and rhamnolipids—from culture supernatant fluids. The ability of surfactant molecules to form micelles at concentrations above the critical micelle concentration allows these aggregates to be retained by relatively high molecular weight cut‐off membranes. Lower molecular weight impurities such as salts, free amino acids, peptides and small proteins are easily removed. Various molecular weight cut‐off membranes were examined for the retention of surfactin and rhamnolipids (mol. wts 1036 and 802 respectively). Amicon XM 50 was the superior membrane for retention of surfactin and a 160‐fold purification was rapidly achieved. The YM 10 membrane was the most appropriate for rhamnolipid recovery. Ultrafiltration can play an important role in biosurfactant purification as large volumes of media can be processed rapidly at extreme