Surface analysis method, mass loss method, and electrochemical test were employed to investigate the corrosion behavior of steel A3 under the effect of Thiobacillus thiooxidans (T.t). Scanning electronic microscopy (SEM) results indicated that the presence of T.t resulted in a more compact biofilm and product film. Pitting occurred on the steel A3 coupon immersed into a sterile system while the one in the T.t system showed general corrosion. The average corrosion rate in the sterile system was greater than that in the T.t system after 3 weeks. Electrochemical impedance spectroscopy (EIS) showed that there were only two time constants for the sample immersed in the T.t system after 10 days. This demonstrates that a compact and strongly inserted product film was formed on the surface of coupon in the presence of T.t which effectively obstructed the erosion of caustic ions. Polarization curve results showed that the corrosion current density of the metal decreased after 20 days of exposure to the T.t solution.