In this work, we present an electrochemical study using a carbon paste electrode modified with chalcopyrite (CuFeS2) in solution A of T&K medium with different ferrous ion concentrations, in the absence and presence of the bacterium Acidithiobacillus ferrooxidans. The aim was to evaluate the influence of ferrous ions and bacteria on the electrochemical behavior of chalcopyrite. Electrochemical impedance spectroscopy (EIS) was used to investigate the processes occurring at the electrode/solution interface in the different systems, considering the charge transfer reactions involving chalcopyrite and ferrous ions, the presence of a multicomponent layer, and diffusion. The main changes in the chalcopyrite response occurred before 67 h or 43 h of immersion, in the absence or presence of ferrous ions, respectively, indicating that the surface oxide layer present on chalcopyrite was dissolved faster in the presence of ferrous ions. The addition of bacteria decreased the charge transfer reaction resistance, especially when ferrous ions were present. In the presence of Fe2+, sulfur and jarosite were detected in the solid residues after leaching, while only jarosite was detected in the bioleaching experiment. The results suggested that ferrous ions accelerated the dissolution of chalcopyrite, and that overlayers including biofilms did not halt chalcopyrite dissolution, indicating that there was no passivation.