Chickpea (Cicer arietinumL.) is an important pulse crop around the globe and a valuable source of protein in the human diet. However, it is highly susceptible to various plant pathogens such as fungi, bacteria, and viruses, which can cause significant damage from the seedling phase until harvest, leading to reduced yields and affecting its production.Botrytis cinereacan cause significant damage to chickpea crops, especially under high humidity and moisture conditions. This fungus can cause grey mould disease, which can lead to wilting, stem and pod rot, and reduced yields. Chickpea plants have developed specific barriers to counteract the harmful effects of this fungus. These barriers include biochemical and structural defences. In this study, the defence responses againstB. cinereawere measured by the quantification of biochemical metabolites such as antioxidant enzymes, malondialdehyde (MDA), proline, glutathione (GSH), H2O2, ascorbic acid (AA) and total phenol in the leaf samples of chickpea genotypes (one accession of wildCicerspecies,viz.Cicer pinnatifidum188 identified with high level of resistance to Botrytis grey mould (BGM) and a cultivar,Cicer arietinumPBG5 susceptible to BGM grown in the greenhouse). Seedlings of both the genotypes were inoculated with (1 × 104spore mL−1) inoculum of isolate 24, race 510 ofB. cinereaand samples were collected after 1, 3, 5, and 7 days post-inoculation (dpi). The enhanced enzymatic activity was observed in the pathogen-inoculated leaf samples as compared to uninoculated (healthy control). Among inoculated genotypes, the resistant one exhibited a significant change in enzymatic activity, total phenolic content, MDA, proline, GSH, H2O2, and AA, compared to the susceptible genotype. The study also examined the isozyme pattern of antioxidant enzymes at various stages ofB. cinereainoculation. Results from scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy revealed that BGM had a more significant impact on susceptible genotypes compared to resistant ones when compared to the control (un-inoculated). In addition, SEM and FTIR spectroscopy analyses confirmed the greater severity of BGM on susceptible genotypes compared to their resistant counterparts. Our results suggest the role of antioxidant enzymes and other metabolites as defence tools and biochemical markers to understand compatible and non-compatible plant-pathogen interactions better. The present investigation will assist future plant breeding programs aimed at developing resistant varieties.