<p>Root rot incited by <i>Macrophomina phaseolina</i> (Tassi) Goid is one of the most destructive diseases of cotton, severely affecting plant growth and productivity. Despite its economic importance, limited information is available on the biochemical and antioxidant defense responses associated with resistance in Asiatic cotton (<i>Gossypium arboreum</i> L.). The present study was undertaken to decipher genotype-specific temporal changes in biochemical constituents and antioxidant enzyme activities in response to <i>M. phaseolina</i> infection. Two cotton genotypes with contrasting reactions to root rot, Garovilli (resistant) and H 174 (susceptible), were evaluated under controlled conditions. The resistant genotype Garovilli consistently accumulated higher levels of total soluble phenolics, sugars, and proteins than the susceptible H 174, suggesting their role in sustaining host defense and metabolism under stress. Antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO), exhibited significantly greater induction in Garovilli, particularly during the early infection stages. This heightened enzymatic activity effectively restricted oxidative stress by scavenging reactive oxygen species (ROS). In contrast, the susceptible genotype H 174 exhibited comparatively lower antioxidant activity and a sharp increase in malondialdehyde (MDA) content, reflecting severe lipid peroxidation and membrane damage. The findings underscore that <i>G. arboreum</i> resistance to <i>M. phaseolina</i> is closely linked to enhanced antioxidant defense machinery and higher accumulation of phenolic compounds, which collectively mitigate pathogen-induced oxidative damage. The distinct biochemical signatures observed between resistant and susceptible genotypes not only provide a mechanistic understanding of cotton root rot resistance but also highlight potential biochemical markers that can be integrated into resistance screening and breeding programs.</p>