The aim of this study was to characterize the chemical composition of some volatile components, in vitro antimicrobial and anticancer activity of essential oil and n-hexane extract from Trachyspermum ammi L. (Family Apiaceae). The chemical composition of samples was obtained by GC-MS analysis, the antimicrobial activity was evaluated by disc diffusion method whereas the in vitro anticancer activity was evaluated by sulphorhodamine method. Twenty-three monoterpenoide compounds were identified in the essential oil in which four compounds; -terpinene, thymol, P-cymene and -pinene were the major components of the oil with quantity 266. 28, 201.97, 194.91 and 38.49 mg/g oil respectively whereas the other nineteen compounds had quantity < 10 mg/g oil. Twelve monoterpene compounds were identified in the n-hexane extract in which three compounds. Thymol, -terpinene and P-cymene were the major components of volatile components of the n-hexane extract with quantity 138.85, 56.41 and 32.69 mg/g extract respectively whereas the other nine compounds had quantity < 10 mg/g extract. The essential oil and n-hexane extract exhibited an antimicrobial activity against five microorganisms and an anticancer activity against HepG2. The essential oil showed higher activity than the n-hexane. Thymol, terpinene and P-cymene of the two samples play an important role in antimicrobial and anticancer activity. In conclusion, this considered the first report that gave the real quantity of each volatile compound in the essential oil and n-hexane extract of T. ammi. Also, this the first work dealing with the anticancer activity of the two samples in addition to the agreement of antimicrobial activity with previous studies. More safety and toxicological studies will need to be addressed if the essential oil and n-hexane extract of T. ammi are to be used for food preservation or medicinal purposes.