Acetylcholinesterase (AChE) is responsible for the rapid hydrolytic degradation of the neurotransmitter acetylcholine into inactive products choline and acetic acid. The purpose of this study was to examine the effect of carbaryl and dichlorvos on the activity of AChE. In this experimental study, 60 samples of free and immobilized form of AChE were prepared. Determination of AChE activity followed the Ellman's method with modifications. Briefly, 200 µl of the enzyme solution was combined with 400 µl of 25 mM phosphate-buffered saline, 200 µl of DTNB [5,5'-dithio-bis(2-nitrobenzoic acid)], and 200 µl of 300 μM acetylthiocholine iodide. Triplicate (1000 µl) samples were transferred to clean 1.5-ml centrifuge tubes, mixed, and held on ice until analysed and the change in absorbance was measured. For inhibition studies, substrate solutions were pre-incubated with dichlorvos and/or carbaryl. Dichlorvos and carbaryl were used at the concentrations of 100 and 500 μM. The activity was evaluated at 412 nm using Ceceil, CE 1020 spectrophotometer. Phosphate buffer (pH 7.35) was used for blanks. AChE activity was quantified as mM/ml/min. AChE activity of free form is more affected by Dichlorvos (0.09 ± 0.03 mM/ml/min) than immobilized form (0.19 ± 0.02 mM/ml/min). AChE activity of free form is more affected by carbaryl (0.11 ± 0.01 mM/ml/min) than immobilized form (0.1 ± 0.04 mM/ml/min). Comparison of mean AChE activity showed that the activity of the enzyme in presence of dichlorvos and carbaryl was significantly lower compared to controls. To calculate the significance of the difference, the t-test for paired values was applied. The results of our study indicate that dichlorvos and carbaryl cause decrease in AChE activity for both free and immobilization form of enzyme. It is therefore concluded that measuring AChE activity is a way to evaluate poisoning with carbaryl and dichlorvos.