The use of biosurfactants derived from bio-based feedstocks which present several advantages over conventional surfactants such as low toxicity, high degradation kinetics, versatility in mineral flotation, it can act as a collector or frother agent. The present work aims to evaluate the use of biosurfactant extracted from bacteria Rhodococcus opacus in the hematite concentration of the iron ore tailings. Firstly, characterization studies of the samples were carried out (granulometric, chemical and mineralogical analysis). Measurements of Zeta Potential, Fourier Transform Infrared Spectroscopy (FTIR) were also carried out to evaluate the interaction of the biosurfactant and sodium silicate on the mineral surface (hematite). The physicochemical properties of biosurfactant were determined by surface tension. Different microflotation studies (Patridge-Smith cell) and batch (CDC mechanical cell) were carried out to evaluate the pH (2, 3, 7, 9, 11), the biosurfactant concentration (1000, 2000, 4000, 6000 and 8000g/t), and the depressor concentration (100, 300, 600, 900 and 1200 g/t) on recovery and Fe grade. In addition, flotation circuit tests (rougher, cleaner, scavenger) were carried out aiming to increase recovery and Fe grade. The critical micellar concentration (CMC) of the biosurfactant was reached at a concentration of 1 g/L. Hematite recovery was possible at pH 3. According to the studies of infrared spectroscopy and zeta potential, there was interaction between the biosurfactant, the sodium silicate on the hematite surface. The Fe recovery and Fe grade in the microflotation was around 37 percent for 6000 g/t biosurfactant concentration at pH 3. The Fe recovery and Fe grade in the batch flotation (Rougher) was approximately 28.50 percent and 44 percent, respectively for 2000 g/t biosurfactant concentration at pH 3, tests were also performed in the presence of sodium silicate (600 g/t) obtaining around 50.50 percent Fe recovery and 58 percent Fe grade. Furthermore, flotation circuit tests (rougher, cleaner and scavenger) were carried out, achieving a Fe recovery and Fe grade around 44 percent and 64.8 percent, respectively. Therefore, the results showed the potential use of the biosurfactant as a collector in the hematite flotation from the iron ore tailings, it may be applied in the mining industry in the future, replacing conventional collectors with the advancement of the studies.