Depending on the importance of the acclimatization step during micropropagation of shrubs, the ex-vitro acclimatization of two azalea cultivars was investigated about the impacts of 50, 100, and 150 µmol m^-2 s^-1 photosynthetic photon flux density (PPFD). To investigate the influence of PPFD on azalea plants during ex vitro acclimatization, the morphological growth traits, photosynthetic indices, antioxidant enzymes, (reactive oxygen species) ROS, and Malondialdehyde (MDA) were studied. However, fresh and dry microshoot weight, plant height, and root length were recorded the better levels when treated with the highest PPFD (150 µmol m^-2 s^-1). Also, microshoots cultivated in the highest levels of PPFD also had the best levels of net photosynthesis rate (NET), chlorophyll, and carotenoid with a decline in Fv/Fm values. Ex-vitro formed leaves had significantly higher pigment (chlorophyll and carotenoids) content than in vitro-grown leaves. Throughout the acclimatization phase, superoxide dismutase (SOD) activity increased. Similarly, increased activity of the enzymes catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) were also noted. These findings show that a level of PPFD at the highest level was appropriate for Rhododendron genus which includes azalea shrubs. The obtained results demonstrated that the capacity of plants to form an enzyme defense system that functions as an antioxidant, protecting them from oxidative stress and limiting the production of free radicals.