This study was focused on the description of interaction between Cu2+ ions and the 1:1 mono- and dirhamnolipid mixtures in the premicellar and aggregated state in water and 20 mM KCl solution at pH 5.5 and 6.0. The critical micelle concentration of biosurfactants was determined conductometrically and by the pH measurements. Hydrodynamic diameter and electrophoretic mobility were determined in micellar solutions using dynamic light scattering and laser Doppler electrophoresis, respectively. The copper immobilization by rhamnolipids, methylglycinediacetic acid (MGDA), and ethylenediaminetetraacetic acid (EDTA) was estimated potentiometrically for the Cu2+ to chelating agent molar ratio from 16:100 to 200:100. The degree of ion binding and the complex stability constant were calculated at a 1:1 metal to chelant molar ratio. The aggregates of rhamnolipids (diameter of 43–89 nm) were negatively charged. Biosurfactants revealed the best chelating activities in premicellar solutions. For all chelants studied the degree of metal binding decreased with the increasing concentration of the systems. The presence of K+ lowered Cu2+ binding by rhamnolipids, but did not modify the complex stability significantly. Immobilization of Cu2+ by biosurfactants did not cause such an increase of acidification as that observed in MGDA and EDTA solutions. Rhamnolipids, even in the aggregated form, can be an alternative for the classic chelating agents.