Cyclospermum leptophyllum is plant species known for its medicinal value and pleasant aroma. The aerial part and plant seeds are traditionally used as food additives as a spice. This study aims to isolate the chemical constituents of essential oil of the aerial part of the plant and study their potential antibacterial activities against some food contaminating bacteria. The essential oil of C. leptophyllum (CSEO) was isolated from aerial parts of the plant species and studied using GC-MS and FTIR techniques. The first four major chemical constituents determined from GC-MS analysis of CSEO (for peak area % â¥ 1.15%) were 2,5-dimethoxy-p-cymene (87.09%), 2-methoxy-1-methyl-4-(1-methylethyl) benzene (3.09%), 2-methoxy-4-methyl-1-(1-methylethyl) benzene (1.71%), and humulene (1.15%). 60%, 30%, 15%, 7.5%, and 3.75% of CSEO solutions were prepared and evaluated for their potential antibacterial activities against six food spoilage pathogenic bacterial strains. Three Gram-positive strains: Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Streptococcus agalactiae (ATCC 12386) and three Gram-negative strains: Escherichia coli (ATCC 25922), Proteus mirabilis (ATCC 35659), and Pseudomonas aeruginosa (ATCC 27853) were used as test microorganisms. Compared to ciprofloxacin, a positive control, the promising antibacterial activity was observed for CSEO against S. aureus at minimum and maximum test solutions as the values of the zone of inhibition diameter (ZID, mm) were recorded as 14.33 ± 0.58 for 3.75% CSEO solution and 30.67 ± 0.58 for 60% CSEO solution. Tests of CSEO solutions generally showed stronger antibacterial activities against Gram-positive than Gram-negative strains. Therefore, CSEO contains potent chemical constituents that might be applicable in treating pathogenic bacterial species.