The binding study of pesticides with proteins is of great importance in ecotoxicology. In this study, a comparative interaction mechanism of phytocystatin with three pesticides has been presented, each from a different class-glyphosate herbicide (GPS), chlorpyrifos insecticide (CPF), and mancozeb fungicide (MCZ). The interaction of purified chickpea cystatin (CPC) has been characterized by fluorescence, UV, and circular dichroism (CD) spectroscopic methods. The study revealed association constants (K_a) of 52 M⁻¹, 1.145 × 10³ M⁻¹, and 36.12 M⁻¹ for the interaction of CPF, MCZ, and GPS with CPC, respectively, signifying the high affinity interaction for MCZ. Structural changes (at tertiary and secondary levels) were confirmed by UV-visible, intrinsic fluorescence and CD spectroscopy. The results showed that the effect on the CPC structure was more pronounced in the case of MCZ, which was followed by CPF and then GPS. The functional analysis of the pesticide treated inhibitor showed a decline in antipapain activity which varied with the time and dose as well as the class of pesticide. MCZ was relatively much more toxic as compared to CPF and GPS. Reactive oxygen species responsible for inhibitor damage were also analyzed. The results obtained implicate that the exposure of plants to pesticides may lead to physicochemical changes in proteins such as phytocystatins leading to physiological damage to the plant system.