An experiment was carried out to study the progression of Fusarium wilt infection and the biochemical reaction of some gladiolus genotypes to Fusarium oxysporum f. sp. gladioli in the root tissue. On infection, there was an oxidative outburst in root tissue that build up a defense mechanism in the plants. The per cent disease index (0–3 scale), histopathological, physiological and biochemical parameters were studied in resistant and susceptible gladiolus genotypes. The percent disease indices were lower in the more resistant genotypes Swarnima and Pusa Unnati compared to the susceptible genotypes Urmi and White Friendship. Total chlorophylls, carotenoids, intercellular CO2, total sugars, total phenols, total soluble proteins, lipid peroxidase, H2O2 generation, superoxide dismutase, catalase, polyphenol oxidase, and phenyl alanine lyase activities were recorded. Histopathological observations showed that genotypes, Swarnima and Unnati were less sensitive to fungus-induced plasmolysis, resulting in a prolonged biotrophic phase and temporarily retarded the disease development. Susceptible genotypes, Urmi and White Friendship showed the onset of the necrotrophic phase at the earliest. Total chlorophylls, carotenoids, intercellular CO2, and total soluble proteins content were decreased by 43.92, 47.36, 63.98 and 24.92%, respectively in the inoculated gladiolus genotypes as compared to non-inoculated ones; whereas total sugars, total phenols, lipid peroxidase activity, H2O2 generation, SOD, catalase and polyphenol oxidase activity were enhanced significantly by 25.91, 38.39, 44.00, 76.02, 76.07, 49.85 and 73.07%, respectively. Changes in concentration of these biochemical attributes led to the activation of defense response against F. oxysporum f. sp. gladioli in gladiolus genotypes.