Environmental pollution is one of the most issues directly or indirectly affecting human life. Heavy metals are considered as the most environmental pollutants affecting plant growth and productivity. Thus, herein the toxicity of copper on in vitro growth and development of two banana commercial cultivars i.e. ‘Grand Nain' and ‘Williams-Zeef' was assessed. Six concentrations of cupric sulfate were used in banana proliferation medium, i.e. 0, 100, 200, 500, 1000 and 1500ppm. Results clearly showed the harmful effect of copper on banana performance reflected by a significant reduction in survival rate, leaf number, fresh and dry weights as well as a complete inhibition of root formation at higher concentrations. Furthermore, excess of copper was able to reduce photosynthetic pigments contents (chlorophyll-A, -B and carotenoids). The two banana cultivars were affected differently under copper levels. In general, ‘Williams-Zeef' was more tolerant than ‘Grand Nain' as exposed by less reduction in both morphological and physiological characteristics. In vitro screening protocol used in this investigation was easy, simple, inexpensive and reduces time and space which are regularly needed to achieve such tasks. With which it was easily to figure out the harmful effect of copper and clearly differentiate between the tested banana cultivars. The results of this study could help in banana improvement by selecting the proper genotypes to be used in copper-contaminated soils, after increasing the number of screened cultivars.