Trace metal pollution is increasing worldwide due to human activities. In this study, we investigated the physiological and biochemical effects of heavy metals on henna plants (Lawsonia inermis L.) grown in oases situated at various distances from an industrial complex in arid southern Tunisia. Our results showed that leaves had higher mean Zn and Cu concentrations than roots, while the reverse was true for Pb and Cd. Such metal immobilization in root cells is related to an exclusion strategy. The biological concentration factor (BCF) of Cu was > 1 at all studied sites, indicating the potential of henna plants to accumulate Cu from the soil. Heavy metals affected various physiological parameters of henna, including water status, macronutrient content, and photosynthetic activity, and increased the rate of hydrogen peroxide (H2O2) production and lipid peroxidation in leaves and roots, indicating oxidative stress. Deleterious heavy metal effects induced biochemical mechanisms in henna plants by accumulating proline and soluble sugars and activated the antioxidant system by enhancing the activities of superoxide dismutase, catalase, and glutathione peroxidase. The magnitude of accumulation and activation was proportional to the concentration of heavy metals in the plant tissues. The stimulation of enzymatic and non-enzymatic antioxidant systems reflects the ability of henna plants to survive in highly polluted environments.