Background and objectives: Variety of extraction methods coupled with definite solvents could increase the removal rate ofmajor constituents from plants. This research has been conducted to evaluate the effect of extraction methods on the main group of compounds, cytotoxicity, anti acetylcholinesterase (AChE) and antioxidant activity of Trachyspermum ammi fruits. Methods: To compare the quality of extracts earned from maceration and reflux techniques, the amounts of total phenolics, flavonoids and antioxidant property of T. ammi’s fruits extracts were determined; moreover, the cytotoxic activity against Human acute lymphoblastic leukemia (ALL) cell lines (NALM-6) was conducted using MTT assay. Anti-acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity of both extracts were also examined by Ellman’s method. Results: The extraction yield of the plant was significantly higher for maceration compared to reflux extraction. Also, both antioxidant activity and total flavonoid contents (IC₅₀=132.95 µg/mL and 140.15 mg catechin/g dry extract, respectively) showed higher amounts considerably in the maceration extraction. In reverse, the content of phenolic compounds (147.28 mg gallic acid/g dry extract and 16.6 mg thymol/g dry extract) was elevated in the refluxed extract. The result exerted moderate inhibition on butyrylcholinesterase activity (IC₅₀₌ 394.161 µg/mL) and cytotoxicity (IC₅₀ =166.92±1.76 μg/mL for NALM-6 cell line) of the extract using maceration. Conclusion: The maceration method could provide additional amounts of major constituents and greater biological properties compared to the reflux technique.