Climate change-driven salinity stress increasingly threatens agricultural production, particularly for salt-sensitive crops like bananas (Musa spp.). Understanding the physiological and elemental responses of banana cultivars to salinity stress is crucial for developing mitigation strategies. This study investigated the in vitro effect of increasing NaCl concentrations (0, 500, 1000, 1500, and 2000 ppm) on the growth, physiological parameters, and element accumulation of banana (Musa acuminata L.) cv. Grandinin cultures. Banana plantlets were exposed to different NaCl concentrations in Murashige and Skooge medium for four weeks. Morphological characteristics, relative water content, chlorophyll content, elemental accumulation (N, K, P, Ca, Mg, Fe, Zn, Mn, Na, and Cl), and Proline content were measured. The results showed that NaCl stress significantly reduced plant growth parameters (shoot and root number, length, and formation percentage) and relative water content. Total chlorophyll content also decreased with increasing salinity. Conversely, Proline content and Na and Cl accumulation in leaves increased. Interestingly, the accumulation of essential elements (N, K, P, Ca, Mg, Fe, Zn, and Mn) decreased with increasing NaCl concentration. In conclusion, In vitro analysis revealed that Musa acuminata L. cv Grandinin exhibits sensitivity to NaCl-induced salinity stress, experiencing reduced growth, altered water balance, and disrupted chlorophyll metabolism. While Proline accumulation indicated a stress response, essential element uptake was compromised at higher salinity levels. These findings suggest the need for further research on salt-tolerant banana cultivars and improved irrigation practices to ensure sustainable banana production in the face of increasing salinity stress.