Acidithiobacillus ferrooxidans has been shown to be a good depressant of pyrite in freshwater and seawater flotation. However, the effect of these bacteria over copper-activated pyrite has not been studied. At the industrial scale, the activation of pyrite with copper is a common process that occurs because Cu2+ ions, released from other minerals, react with pyrite. This is a problem because Cu2+ ions facilitate the reaction of pyrite with the xanthate collectors, becoming hydrophobic and reaching the froth. In this study, microflotation experiments in a Hallimond tube were conducted to evaluate the depressant effect of A. ferrooxidans over non-activated and Cu-activated pyrite in freshwater and seawater flotation. The experiments were carried out at pH 4, 6, 8, 10 and 12 and pyrite was mixed with CuSO4 at 2.5×10−5 and 5×10−5 M in order to activate its surface. Considering the results obtained in the microflotation tests, it is possible to conclude that Acidithiobacillus ferrooxidans is able to depress non-activated and Cu-activated pyrite at the entire pH range studied (4–12) in freshwater. On the other hand, the use of bacteria in flotation with seawater proved to be effective to depress non-activated and Cu-activated pyrite at pH 8 and 10 with better results achieved at pH 10. At this pH, the non-activated pyrite recovery dropped from 96% to 15%, and the recovery of Cu-activated pyrite dropped from 95% to 32% when the activation was carried out at 2.5×10−5 M, and from 87% to 50% when the activation was conducted at 5×10−5 M of CuSO4. The XPS analysis showed that chalcopyrite and copper (II) hydroxide were formed on the pyrite surface when it is contacted with CuSO4.