In vitro growing of Melaleuca can contribute to the cloning of superior genotypes. Studies on factors affecting micropropagation are necessary, especially those using recent technologies, such as light-emitting diodes (LEDs). This study aimed at better understanding the effects of wavelengths on the development and physiology of Melaleuca cultivated in vitro, as well as the interaction of LED with N6-Benzylaminopurine (BAP), the main cytokinin used in micropropagation. The BAP and wavelengths, and their interaction, had a strong influence on most variables analyzed, altering the in vitro development and chlorophyll concentrations in the plants and changing the different variables of the culture medium, such as pH, EC, levels of Ca2+, Mg2+ and P in culture media and the nutrient accumulation in the shoots. The results demonstrated that the main effects of adding BAP in the in vitro cultivation of Melaleuca are the increase in the number of shoots, which resulted in a greater increase in fresh and dry mass, reduction in height and chlorophyll contents, complete inhibition of adventitious rooting, higher consumption of Mg and lower consumption of Ca and P from the culture media, higher contents of Fe and lower contents of P, S, Mn, Cu and B in the in vitro tissues.