Background: Leaf blight caused by Alternaria spp. is one of the serious concerns in guar production worldwide. Despite this, the interaction of host-pathogen and responses of different guar genotypes against necrotrophic fungus Alternaria is still not much explored. Methods: In the present investigation, six guar genotypes have been infected with Alternaria and observed differential responses by using various parameters, including biochemical assays, antioxidant activities (SOD, CAT, PO, GPx, APx PAL, β-1,3-glucanase enzyme, total phenolic content) and physiological parameters (chlorophyll content, carotenoid and photosynthetic yield-II). Result: After the pathogenicity of Alternaria there was a substantial shift in the biochemical activity in all the inoculated resistant genotypes compared to their respective controls. Moreover, a significant reduction was also observed up to 65% in terms of physiological performance, such as total chlorophyll content, carotenoids and photosynthetic yield-II content, after 14 days of pathogenicity. The genotypes RGC-1066 and RGC-936 showed relatively more resistance and RGC-936 showed relatively more susceptibility towards Alternaria infection. These findings also imply that host-pathogen interactions factors have a role in the differential expression of defence-related enzymes and antioxidant activities in response to Alternaria inoculation.