The effect of temperature on nickel sulfide bioleaching was studied in the presence of mesophile (Acidithiobacillus ferrooxidans) and moderate thermophile (Sulfobacillus thermosulfidooxidans) strains and the results were discussed in terms of sulfide dissolution thermodynamics (Eh–pH diagrams) and kinetics (cyclic voltammetry). It was observed that in the pH range 1.8–2.0 the highest nickel dissolution was achieved which reached 50% for mesophiles and over 80% for moderate thermophiles. External ferrous iron addition had no effect on the metal dissolution at 34 °C, but adversely affected nickel leaching at higher temperatures. The best outcomes were accomplished with low FeSO4 additions (2.5 g/L) at 50 °C. Pyrrhotite dissolution avoided the need for external iron addition, providing Fe2+ concentrations as high as 7 g/L during bioleaching, which supports bacterial growth. Eh–pH diagrams for pentlandite and pyrrhotite show a negligible effect of temperature on the stability field of each sulfide whilst cyclic voltammetry indicated that temperature has the strongest influence on pyrrhotite oxidation. The latter along with a rapid increase in solution potential (Eh) explains the higher and faster extraction observed with S. thermosulfidooxidans.