The present study was carried out to investigate the effect of physostigmine (a carbamate pesticide) on the electrophoretic patterns of esterase activity in parotoid gland secretion and its extract of common Indian toad Bufo melanostictus (Schneider). The patterns of the esterases were examined on 7.5% of polyacrylamide gel electrophoresis (PAGE) stained with α- napthyl acetate. The parotoid gland secretion and its extract were exposed to the toxicant physostigmine and the variations were observed on the activity of esterase banding patterns at different time intervals i.e., 4 hours, 8hours, and 12 hours. The findings that the parotoid gland extract showed homology in esterase bands with more intensity compared to than the parotoid gland secretion.