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The objective of this study was to investigate the alterations in physiological, biochemical, and transcriptional reactions in the flag leaf of rice following the application of Trichoderma as a biofertilizer (BF) at both ambient (aCO2; 395 ppm) and enhanced (eCO2; 550 ppm) carbon dioxide levels. A comparatively smaller percentage change was observed in the photosynthetic parameters, including photosynthetic rate, respiration, and stomatal conductance, subsequent to the introduction of BF in the elevated carbon dioxide (eCO2) environment. Furthermore, biochemical analysis revealed a noteworthy elevation in photosynthetic pigments, total sugar, proline, and ascorbate peroxidase concentration levels in plants treated with BF under ambient and increased carbon dioxide conditions. The buildup of indole‐3‐acetic acid (IAA) was shown to be increased in rice treated with BF under both conditions. Moreover, ICP‐MS analysis demonstrated an augmentation in the levels of vital micronutrients in the experimental group while observing no alterations in the carbon‐to‐nitrogen ratio within the flag leaf. The examination of transcriptional responses in the flag leaf demonstrated an increase in the expression of genes associated with the photosynthetic system, amino acid metabolism, several transporters, including aquaporin, and the phosphate pathway, as well as carbon and nitrogen metabolism. Hence, our work elucidates the effective role of Trichoderma in mitigating the adverse consequences associated with elevated levels of atmospheric carbon dioxide (eCO2). [ABSTRACT FROM AUTHOR]