The effect of a rhamnolipid biosurfactant produced by a Pseudomonas aeruginosa MA01 strain on desulfurization of iron concentrates was studied. Surface tension measurement and frothing characterization indicated better surface activity and frothability of rhamnolipid compared to methyl isobutyl carbinol (MIBC) as an operating frother. Reverse flotation tests using rhamnolipid either as a sole frother or mixed with MIBC, showed that the desulfurization process is more efficient at pH 4.5 and high concentration of rhamnolipid in the presence of MIBC. However, under these conditions water recovery decreased due to the change in rhamnolipid aggregates morphology. Results from the present study seemed promising to introduce the biosurfactant from Pseudomonas aeruginosa as a new frother.