Anaerobic nitrate-dependent iron(II) oxidation is a process common to many bacterial species, which promotes the formation of Fe(III) minerals that can influence the fate of soil and groundwater pollutants, such as arsenic. Herein, we investigated simultaneous nitrate-dependent Fe(II) and As(III) oxidation by Acidovorax sp. strain ST3 with the aim of studying the Fe biominerals formed, their As immobilization capabilities and the metabolic effect on cells. X-ray powder diffraction (XRD) and scanning transmission electron microscopy (STEM) nanodiffraction were applied for biomineral characterization in bulk and at the nanoscale, respectively. NanoSIMS (nanoscale secondary ion mass spectrometry) was used to map the intra and extracellular As and Fe distribution at the single-cell level and to trace metabolically active cells, by incorporation of a 13C-labeled substrate (acetate). Metabolic heterogeneity among bacterial cells was detected, with periplasmic Fe mineral encrustation deleterious to cell metabolism. Interestingly, Fe and As were not co-localized in all cells, indicating delocalized sites of As(III) and Fe(II) oxidation. The Fe(III) minerals lepidocrocite and goethite were identified in XRD, although only lepidocrocite was identified via STEM nanodiffraction. Extracellular amorphous nanoparticles were formed earlier and retained more As(III/V) than crystalline “flakes” of lepidocrocite, indicating that longer incubation periods promote the formation of more crystalline minerals with lower As retention capabilities. Thus, the addition of nitrate promotes Fe(II) oxidation and formation of Fe(III) biominerals by ST3 cells which retain As(III/V), and although this process was metabolically detrimental to some cells, it warrants further examination as a viable mechanism for As removal in anoxic environments by biostimulation with nitrate.