Fe(III) oxyhydroxides play critical roles in arsenic immobilization due to their strong surface affinity for arsenic. However, the role of bacteria in Fe(II) oxidation and the subsequent immobilization of arsenic has not been thoroughly investigated to date, especially under the micro-oxic conditions present in soils and sediments where these microorganisms thrive. In the present study, we used gel-stabilized gradient systems to investigate arsenic immobilization during microaerophilic microbial Fe(II) oxidation and Fe(III) oxyhydroxide formation. The removal and immobilization of dissolved As(III) and As(V) proceeded via the formation of biogenic Fe(III) oxyhydroxides through microbial Fe(II) oxidation. After 30 days of incubation, the concentration of dissolved arsenic decreased from 600 to 4.8 μg L⁻¹. When an Fe(III) oxyhydroxide formed in the presence of As(III), most of the arsenic ultimately was found as As(V), indicating that As(III) oxidation accompanied arsenic immobilization. The structure of the microbial community in As(III) incubations was highly differentiated with respect to the As(V)-bearing ending incubations. The As(III)-containing incubations contained the arsenite oxidase gene, suggesting the potential for microbially mediated As(III) oxidation. The findings of the present study suggest that As(III) immobilization can occur in micro-oxic environments after microbial Fe(II) oxidation and biogenic Fe(III) oxyhydroxide formation via the direct microbial oxidation of As(III) to As(V). This study demonstrates that microbial Fe(II) and As(III) oxidation are important geochemical processes for arsenic immobilization in micro-oxic soils and sediments.