Rhamnolipids have received great attention in various environmental applications in terms of metal complexation and recovery. However, the influence of metal ions on the interfacial, foaming, and ion flotation properties of rhamnolipid are poorly investigated. In this study we investigated the effect of metal ions alone and in a mixed metal system on the interfacial and foaming properties of rhamnolipid. Further, the potential of rhamnolipid to recover and separate Gallium from a mixed metal system containing Gallium (Ga) and Arsenic (As) using bioionflotation has been investigated. The effect of operating parameters like pH, rhamnolipid concentration, and airflow rate were tested and found to have a significant influence on the separation performance. The maximum removal of Ga could reach 74 % when rhamnolipid concentration was 0.85 mM at pH 6 and an airflow rate of 80 ml/min. The selectivity index of Ga over As was highest (17.2) at 0.85 mM rhamnolipid concentration, pH 6, and an airflow rate of 40 ml/min. Also, the selective separation of Ga was dependent on the recovery of water from the foam. The results showed that rhamnolipid biosurfactant acted as a highly efficient ion collector for Ga and the optimized process parameters could be expected to provide very efficient separation and recovery of target metal via ion flotation.