The interaction between different bioreagents and minerals has been studied for many years. Rhodococcus opacus and Rhodococcus erythropolis biosurfactants can behave as collectors and frothers in mineral flotation. This work aims to evaluate the interaction between them mixed in different percentages for recovering hematite from an iron ore tailing composed of low grade of hematite and quartz as gangue. The interaction of these two biosurfactants was investigated using different techniques such surface tension, zeta potential, contact angle (Ɵc), and Fourier Transform Infrared (FTIR) to understand the behaviour at the different interfaces. The surface tension analyses of each biosurfactant in contact with water reached the lowest value in 32.35 mN.m-1 (Rhodococcus opacus) and 31.41 mN.m-1 (Rhodococcus erythropolis) at acidic conditions pH 3. The experimental zeta potential measurements revealed the positive interaction of both biosurfactants on the hematite surface. The isoelectric point (IEP) of hematite was near pH 3.40 and the charge reversal point of Rhodococcus opacus and Rhodococcus erythropolis was achieved in pH 2.20 and pH 3.30 respectively. The IEP of quartz was around pH 2.12 and the zeta potential decreased when both biosurfactants were added, it was not possible to get the displacement of the IEP after interaction with them. The FTIR analyses confirmed the affinity of hematite surface with both biosurfactants, the opposite happens in the case of quartz, there was weak adsorption on the surface. Finally, the Ɵc of hematite 40 ° after interaction with R. opacus biosurfactant increased to 64° and with R. erythropolis biosurfactant amplified to 87°. This changes indicate a good hydrophobization of hemtatite. The results of microflotation tests in the Partridge-Smith modified cell revealed that a huge percent proportion of R. erythropolis led to an increase in the metallurgic recovery of about 48.07% and 24.35 % of iron concentrate.