Bioleaching is a process that involves acidophilic and chemolithoautotrophic microorganisms such as Acidithiobacillus ferrooxidans. It is known that At. ferrooxidans undergoes growth and biooxidation inhibition due to ferric ion accumulation. The objective of this study was to establish a strategy to adapt planktonic cells of At. ferrooxidans to high total iron concentrations in two steps and correlate their tolerance to ferric ion with extracellular polymeric substances (EPS) accumulated on the cell surfaces. The first step consisted in carrying out pulse additions of ferrous ion and the second one comprised consecutive sub-cultures with the objective of improving the biooxidation capacity of the bacteria at different concentrations of total iron (18, 27 and 36 g L−1). Visualization and quantification of EPS were assessed through confocal laser scanning microscopy (CLSM). The adapted cells exhibited an increasing volumetric productivity of ferric ion and cell density according to the total iron concentration when adapted and superior to what was showed by non-adapted cells of the control cultures. It was observed that cells adapted to higher total iron concentration displayed the greatest amount of EPS accumulated on their surfaces. Maximum tolerance to total iron of cells adapted to 36 g L−1 was 20% greater than non-adapted cells of the control culture. The experimental evidence points out that EPS accumulation by planktonic At. ferrooxidans was a determining factor for its tolerance to high iron concentrations. Moreover, it is thought that these results could be important in order to increase the efficiency in processes such as bioleaching and biooxidation.