Both ex vitro and in vitro propagation techniques play crucial roles in banana production. However, there is limited knowledge on the comparison of physiological functions between plantlets derived from these methods. The assessment of physiological functions in plantlets during the nursery stage holds significant importance, as it provides a comprehensive understanding of their responses to growth conditions. Therefore, this study aimed to estimate the levels of photosynthetic pigments, including chlorophyll a, b, total chlorophyll, and carotenoids, as well as evaluate the activity of defense enzymes such as superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POX) in ex vitro and in vitro propagated plantlets of five distinct banana cultivars, namely Red Banana (AAA), Nendran (AAB), Rasthali (AAB), Ney Poovan (AA), and Monthan (ABB). The results demonstrated significant variations in both photosynthetic pigments and antioxidant defense enzyme activities between the two propagation methods. Ex vitro propagated plantlets exhibited higher levels of photosynthetic pigments and defense enzymes in compared to in vitro propagated plants. These results support ex vitro propagation to produce physiologically active planting material in assessed banana cultivars.