The present study aimed to investigate the effects of zinc exposure (0, 1, 5, 15, 30 mg/L zinc) on the biochemical and physiological parameters of Peganum harmala seedlings. Two populations (metallicolous and non-metallicolous) were compared in Zn tolerance, Zn accumulation, photosynthetic pigments, and enzymatic antioxidant activities. Plants were treated with Zn at concentrations of 0, 1, 5, 15, and 30 mg/L for 14 days. The study results showed that the increase of Zn concentration in the nutrient solution reduced shoot length, root length, root dry weight, shoot dry weight, chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid in both populations; however, the accumulation was more pronounced in metallicolous populations (M) than in non-metallicolous (NM) ones. In response, the activities of antioxidant enzymes such as guaiacol peroxidase, lipoxygenase, superoxide dismutase, catalase, and ascorbate peroxidase were enhanced Zn exposure in both populations. Moreover, it was found that the metallicolous population of P. harmala had a greater capacity to adapt to oxidative stress caused by Zn than the non-metallicolous population, and antioxidative defense in the metallicolous population of P. harmala might have played an essential role in Zn tolerance. Therefore, P. harmala seemed to be a suitable candidate for accumulation; however, it was recommended that further investigations be carried out to explore its metal remediation ability. It is concluded that P. harmala can be a potential candidate for bioremediation of Zn contaminated soils.