Pimpinella anisum L. (family Apiaceae) is widely used for curing variety of ailments. The objective of this studywas to evaluate the antimicrobial activity and chemical composition of chloroform and ethanol extracts from the seed of Pimpinella anisum against standard microorganism. This plant has been used as a traditional treatment for several diseases such as microbial infections. Extracts were evaluated for their effectiveness against four bacterial strains including both Gram positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa)bacteria as well as fungal species (Candida albicans and Aspergillus niger) using disc diffusion method. The chloroform extract showed the highest activity against B.subtilis (11 mm), E. coli (16mm) and P. aeruginosa (14.5 mm) but the same result against S. aureus in two extracts (15.5 mm). All extracts exhibited high antifungal activity against C. albicans and A. niger with inhibition zone ranging from (14.5 to 20 mm). The quantitative analysis of chemical composition determined by Gas Chromatography–Mass Spectrometry (GC-MS). The result showed high amounts of Butanoic acid, 2-methyl-, 2-methoxy-4-(2-propenyl) phenyl ester (23.21%) and Anethole (12.52%).