Ajwain (Trachyspermum ammi) is a source of medicinally active compounds and has various pharmacological effects and therapeutic uses. The aim of this study was to evaluate the antimicrobial activities of ajwain seeds ethanolic extract against different pathogenic bacteria. Ethanolic extraction of ajwain seeds was carried out by supercritical fluid extraction method and GC-MS of the extract showed the presence of thymol as its major component (71.06%) along with o-Cymene (3.37%), γ-Terpinene (3.83%), 2-methyl-5-(1-methylethyl)-phenol (0.51%). The extract was analyzed for its ethanolic DPPH (2, 2-diphenyl-1-picrylhydrazyl) scavenging potential, showing increase in percent inhibition (50% to 83%) with increase in concentration (50 µg/mL to 250 µg/mL). The antimicrobial test results revealed that the pure ethanolic extract of ajwain seeds is significantly active against the food-borne pathogenic Gram positive and Gram negative bacterial strains (range of inhibition, 15-19 mm) and was compared with that of the broad-spectrum antibiotic, chloramphenicol (range of inhibition, 33-37 mm). Results concluded that ajwain seeds contain high amount of phenolic compound (mainly thymol) and have high antioxidant and antimicrobial activity and, hence, can be used as an excellent biopreservative and for medicinal purpose, and can be useful for various perishable and fat rich food products by enhancing their shelf-life.