The present research work studies the effect of rhamnolipid biosurfactant (RL) produced from Pseudomonas aeruginosa bacteria on the metallurgical response of a copper ore sample flotation through an extensive full factorial experimental design. Key influential factors including feed particle size, pulp solid content, pH, and dosages of collector, frother and RL biosurfactant were considered. The surface activity of the RL biosurfactant was also studied based on a D-optimal experimental design. Surface activity results revealed that increasing pH and electrolyte concentrations negatively impacted the RL surface activity, while the effect of electrolyte source was dependent on their ionic strength. Metallurgical investigations showed that operating parameters significantly influence the copper grade and recovery with considerable interaction among various parameters. RL biosurfactant was found to negatively decrease the copper grade (~0.5%) and positively enhance the recovery (~3%). Effect of RL was attributed to two potential mechanisms, i.e., being ineffective on copper minerals and/or interaction with gangue minerals, as well as increasing the rate of entrainment due to high foamability, both of which increased non-selective recovery of gangue minerals. Interestingly, regardless of the structural similarities, no interaction between the flotation reagents and rhamnolipid was observed. Fourier-transform infrared (FTIR) spectroscopic analysis of copper minerals, both pure and RL-exposed, showed that there was actually no molecular interaction between RL molecules and particle surface.