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The contamination of abandoned mining areas is a problem worldwide that needs urgent attention. Phytoremediation emerges as a successful method to extract different contaminants from the soil. In this context, Eucalyptus globulus plants growing in soils artificial contaminated with arsenic (As) were used to access its phytoremediation capabilities. The effects of As on photosynthetic performance were monitored through different physiological parameters, whereas the uptake and translocation of As and the putative effects on calcium, iron, potassium, and zinc levels on plants were evaluated by X-ray fluorescence analysis. Root system is the major accumulator organ, while the translocation to the above-ground organs is poor. In the end of the experiment, the root biomass of plants treated with 200 μg As mL−1 is 27% and 49.7% lower than equivalent biomass from plants treated with 100 μg As mL−1 and control plants, respectively. Each plant can accumulate 8.19 and 8.91 mg As after a 6-month period, when submitted to 100 As and 200 As, respectively. It seems to exist an antagonistic effect of As on Zn root uptake by E. globulus. In general, the tested concentrations do not influence negatively plant metabolism, indicating that this species is suitable for plantation in contaminated areas.