In most non-hyperaccumulating plants, Ni and Zn uptake is negatively correlated with soil pH, however, few studies so far have investigated how pH influences the activity and uptake of Ni and Zn in plants grown in a hydroponic system, which generally allows culture variables to be singularly manipulated. In this study, the non-accumulator Stellaria media (L.) Vill. (Caryophyllaceae) had opposite trends of Ni and Zn uptake along a pH gradient (between 5 and 8 for Zn and between 5 and 6.5 for Ni), when grown in hydroponics. In all treatments, the solution metal concentration was fixed at 0.1 mM Ni or 0.55 mM Zn. Nickel accumulation increased with increasing pH with an average concentration in shoots of 167 μg/gDW at pH 5 and of 250 μg/gDW at pH 6.5. In contrast, Zn accumulation decreased with increasing pH, with an average concentration in shoots varying from 1640 μg/gDW, at pH 5, to 435 μg/gDW at pH 8. Assessment of total polyphenol and flavonoid contents and of antioxidant activity showed that these parameters were positively correlated with Ni or Zn accumulation in S. media shoots, while photosynthetic pigments content and root and shoot biomass were negatively correlated with Ni and Zn accumulation. The study was carried out on five different S. media populations, which did not show differences in relation to the accumulation of metals and synthesis of antioxidant compounds, nonetheless showing a different biomass production under control conditions.