In many places around the world, arsenic is considered a primary pollutant in water due to its high toxicity. Various methods have been developed for the treatment of arsenic contaminated soil and water. In this study, the efficiency of high lactonic sophorolipids (SL18), in removing arsenic and heavy metals from mine tailings through the process of soil washing, was evaluated. Sophorolipids are capable of increasing the solubility of organic compounds and binding metalloids and heavy metals and are biocompatible, rapidly biodegradable, and non-toxic. To investigate the efficiency of sophorolipids in heavy metal/metalloid removal, a series of batch and column tests, using different concentrations of sophorolipids (0.1, 0.5, 1, 2, 3, 4 and 5%), at different pH levels (2, 4, 6, 8, 10 and 12), and at three different temperatures (15ºC, 23ºC and 35ºC) was performed. Furthermore, the effect of sophorolipids on the speciation of arsenic, and the effectiveness of sophorolipids on different fractions of the mine tailings were examined. The results of this investigation show that using a 1% sophorolipid solution in a column test resulted in the removal of 83% of the arsenic. To identify the mechanism of removal of arsenic from mine tailings, the effect of sophorolipids on mine tailing sample through a sequential extraction procedure combined with Scanning Electron Microscopy (SEM), X-ray powder diffraction (XRD) scans was investigated. Also, the spectrum of sophorolipids was studied using Fourier-transform infrared spectroscopy (FTIR) and the effect of the presence of arsenic and iron on its functional groups was determined. Dynamic light scattering (DLS) measurements were used to find the effect of pH and mine tailing constituents on the size of micelles. Investigations on the mechanism of sophorolipid assisted arsenic removal confirmed that the formation of arsenic complexes with sophorolipid functional groups, reducing the interfacial tension and solubilization, changing the net charge of surfaces and ion exchange are the main mechanisms. The impact of each mechanism is chiefly governed by the pH. The results from this study shed light on the mechanism of arsenic removal by biosurfactants and can aid in the development of a sustainable and environmentally friendly solution for mine tailing remediation.