ZnO is extensively used in a wide range of industrial applications. Hence, considerable accumulation of this substance occurs in the environment. The objective of the present study was to compare and characterize the growth of model tobacco plant at different ZnO levels in various rooting media (i.e., water-based, perlite-based, and sand-based). In soilless medium (especially water-based), ZnO levels lower and/or higher than 1μM showed negative impacts on fresh weights, some of leaves indices, and number of flowers and fruits. In soil-based media, 250 and 500 mg ZnO kg-1 often scored the highest values for the mentioned indices. However, all evaluated indices were much higher in water-based than the other two media. Flavonoids, antocyanins and FRAP capacity increased at highest levels in soil-grown and perlite-grown media, but Zn content was the same for all concentrations in soil-grown plants. Photosynthetic pigments decreased at 1,000 mg kg-1 in soil-based media. Overall, the sensitivity to small changes in ZnO levels was much higher in water-based compared to the other two media, while ZnO supply resulted in improvement of some parameters in soil-based media. Soil and perlite possess certain experimental limitations (e.g., surface absorption, unfavorable pH, low gas exchange, limited spread of roots and insoluble Zn-complexes), while water-grown plants were comparatively better than the other media in terms of experimental control and handling. These results show different effects of ZnO levels in different media and also suggest the water-based medium as a possible alternate for future accurate investigations of Zn trials.