With the scarcity of good quality water, plants like tomatoes will be more susceptible to excess boron (EB) in Mediterranean regions. The effects of EB on the growth, free, semi-bound, and bound boron (B) concentrations, and macromolecules of the Solanum lycopersicum L. cultivar Castle Rock, were investigated in this study. Seedlings were exposed to four levels of EB using boric acid. The results manifested that EB inhibited tomato growth, total water content, and photosynthetic pigments. EB harmed the membrane stability, as seen by increased potassium (K) leakage, UV absorbance metabolites, and electrolyte conductivity (EC) in leaf disc solution. EB raised concentrations of free, semi-bound, and bound forms of B in seedlings. Fourier transform infrared spectroscopy (FTIR) data revealed that EB induced uneven wax deposition, altered the shape of cell walls, and lowered cellulose synthesis in seedlings. EB affected the amide I and amide II indicating damage to the protein pools. These results provide new insights into understanding the specific effects of EB on the functional groups of different macromolecules of tomato seedlings.