Climatic changes affect agriculture and alter cultivated land area worldwide. Among which, low-temperature has a general negative impact on plant growth and productivity especially on tropical and subtropical species. To assess the effect of cold stress on banana, two global cultivated varieties (i.e. Grand Nain and Williams) were used for in vitro physio-biochemical evaluation. Cold stress was performed at constant 5 °C for 6, 12, 24 and 48 h, to assess the changes in the pool of proline, total phenolics, total soluble carbohydrates, K+ and Ca+ 2 ions as well as the photosynthesis related pigments chlorophyll and carotenoids). Significant differences between cultivars and among exposure times were found in most of the studied traits. Results indicated that cold-stressed plants were capable to enhance their cold tolerance by over-accumulation of cryoprotectants, particularly under extended cold stress. Importantly, Williams showed more tolerance than Grand Nain by accumulating higher amounts of total phenolics, total soluble carbohydrates, higher concentrations of K+ and Ca+ 2 ions as well as more content of the photosynthetic pigments, compared to Grand Nain. Meanwhile, Grand Nain produced greater amounts of proline than Williams. These findings suggest that compatible solutes increased in relation to cold tolerance mechanisms which modulate chilling-induced oxidative damage in banana. These imperative mechanisms could be utilized as important tools and selectable markers for cold tolerance screening in banana genotypes which could be helpful in breeding programs.