Arsenopyrite (FeAsS) is one of the most typical As-bearing sulfide minerals and bio-oxidation of arsenopyrite is a process of particular scientific interest, due to its broad application in gold mineral processing as well as the release of toxic As. This study aimed to enhance the dissolution of arsenopyrite (as well as of Fe(III)/As-bearing oxidation products) via a sequential two-step process comprising aerobic bio-oxidation and reductive dissolution under anaerobic conditions, both catalyzed by Acidithiobacillus (At.) thiooxidans DSM 504: (i) during aerobic bio-oxidation, 67.7 and 64.9% As was extracted from arsenopyrite in a basal salts medium (BSM) and acid mine drainage (AMD) respectively. A double passivation layer (comprising jarosite and a dense outer layer) formed on the mineral surface in BSM, inhibiting further bio-oxidation, while an oxidation layer with a more permeable amorphous nanosheet structure (composing of tooeleite) was formed in AMD. (ii) Subsequently, the reductive dissolution process effectively eliminated the double passivation layer (composed of S0, amorphous ferric arsenate, K-jarosite, tooeleite, and scorodite), with 47.2 and 91.9% Fe reduced in 16 days of anaerobic bio-oxidation of S0 and S2O32-, respectively. The proposed two-step bio-oxidation process has the potential to contribute to the development of improved extractive technologies, and avoid the accumulation of unstable As pollution sources. Additionally, the data indicate that a replacement of growth media with AMD could reduce the elevated costs of the technology capable of alternating aerated and anoxic bio-oxidation stages.