Aluminum (Al) toxicity poses a significant challenge to agricultural productivity, particularly in acidic soils. The banana crop, predominantly cultivated in tropical and subtropical climates, often grapples with low pH and Al toxicity. This study seeks to explore the differential responses of two banana genotypes with varying Al tolerance (Baodao and Baxi) to Al exposure (100 and 500 µM) for 24 h. Microscopic analysis uncovered distinctive structural modifications in root cells, with Baodao displaying more severe alterations in response to Al stress. There was higher superoxide (O2-.) and hydrogen peroxide (H2O2) production and lipid peroxidation in Baodao indicating enhanced oxidative stress and membrane damage. Al accumulation in root tips was higher in Baxi than Baodao, while the roots of Baodao had a higher accumulation of callose. Nutrient content analysis revealed alterations in ion levels, highlighting the impact of Al exposure on nutrient uptake and homeostasis. In summary, Al differentially affects callose deposition, which, in turn, leads to Al uptake and nutrient homeostasis alteration in two contrasting banana genotypes. This intricate interplay is a key factor in understanding plant responses to aluminum toxicity and can inform strategies for crop improvement and soil management in aluminum-stressed environments.