Cadmium (Cd) is a widespread and strongly toxic environmental pollutant. In this study, the interaction between Cd and essential nutritional metals, such as iron (Fe) and zinc (Zn), was investigated in banana plants (Musa spp. cultivar Grand Nain), cultured in vitro, using Fourier-transform infrared (FT-IR) and physiological analysis. Plantlets were treated in vitro with Fe and Zn (200 and 500 mg/L) under 500 mg/L Cd exposure. The results showed that Cd toxicity increased Cd uptake and raised% of damage. However, Fe and Zn addition ameliorated the negative impact of Cd stress by reducing Cd and enhancing Fe, Zn, P, and K contents. The FT-IR analysis showed alterations within the bands correlated to the foremost macromolecules in plants under Cd stress and its interactions with Fe or Zn. The peaks of some functional groups at 3381.7 cm− 1 for carbohydrates, proteins, alcohols, and phenolic compounds