The use of a biosurfactant (BS) in mineral flotation offers numerous advantages over conventional surfactants, such as their low toxicity, high degradation kinetics, and potential for selectively treating low-grade ores. In the present study, the use of a biosurfactant obtained from Rhodococcus opacus bacteria for the flotation of hematite from iron ore tailings was evaluated. The microflotation assessments were conducted in a modified Partridge-Smith cell, and the batch flotation studies were conducted in a mechanical cell (CDC–cell). In addition, the effects of the pH, biosurfactant concentration, and depressant concentration on hematite recovery were evaluated. The results confirmed the biosurfactant adsorption onto the hematite surface, and the biosurfactant decreased the surface tension of the water/gas interface. The critical micelle concentration (CMC) of the biosurfactant was approximately 1 g.L−1. Hematite recovery was feasible at a pH of around 3. In microflotation tests, the iron grade and recovery reached approximately 37% and 30%, respectively. These values increased in batch flotation circuits, specifically in the cleaner stage, the iron grade reached approximately 44% and the recovery was approximately 65%. Thus, the current development proved that this particular treatment of ore tailings carries environmental and technical benefits as an appropriate alternative cleaning technology.