Electroflotation is a clean technique potentially able to recover fine particles from mineral suspensions. The aim of the present work was to evaluate the electroflotation of fines and ultrafine particles of an itabiritic iron ore using a biosurfactant extracted from Rhodococcus opacus bacteria. Infrared spectroscopy and zeta potential measurements confirmed the interaction between the biosurfactant and the mineral surface. The isoelectric point of hematite presented a value of about pH 5.3; after interacting with the biosurfactant, a charge reversal point of pH 3.5 was observed. The biosurfactant reduced the air/water surface tension from 71 to 40 mN/m, using 25 mg/L concentration. The electroflotation process of fine and ultrafine particles was evaluated as a function of pH, biosurfactant concentration, stirring of the aqueous suspension and current density. It was observed that the iron recovery (%) and iron grade (%) were negatively affected by increasing pH value. Therefore, best results were achieved at pH 3. Biosurfactant concentration and current density positively affected both response variables. An iron recovery value of about 83% and an iron grade of about 59% were achieved for the −38 + 20 µm size fraction; whereas, higher values were attained (98% and 64%, respectively) for the finer size fraction −20 µm.