Purpose: Arsenic (As) is one of the most widespread toxic elements, affecting human health through consumption of contaminated water or food. This work studied the effects of ammonium sulphate (NH4)2SO4 on mobility, speciation, and toxicity of As in a soil polluted by the destruction of chemical ammunition. Methods: Effects of fertilization on mobility and speciation of As, abundance of active AsIII-oxidizing and AsV-reducing microorganisms, abundance of bacterial 16S rRNA genes, and aioA and arsB genes involved in As biotransformation were monitored during barley growth in polluted (P) and non-polluted (NP) soils in pots maintained in outdoor conditions, with either the usual dose of (NH4)2SO4, 10 × or 100 × this dose. As transfer to plants and phytotoxicity were evaluated through quantification and speciation of As in grains and lipid peroxidation in plant leaves. Results: As mobility was significantly reduced by the highest dose of (NH4)2SO4. Fertilization tended to increase the Omega-3 Index in barley with the P soil. Abundance of AsIII-oxidizing microorganisms was higher and plant biomass was lower in P than in NP soil. Neither pollution level nor fertilization dose affected As speciation in soils or gene abundance. The sum of the concentrations of extracted AsIII and AsV species in grains was in the range 0–150 µg kg−1 with NP soil and 2000–3500 µg kg−1 with P soil. Conclusions: Results underline the importance of developing surveys of As dynamics in agricultural contexts with diverse types of crops, with a focus on As speciation mechanisms, linked to soil microbial activities and plant physiology.< Réduire