This study is to investigate the antibacterial activity of the alcoholic and hydroalcoholic extracts of Spinacia oleracea leaves, on Listeria monocytogenes (ATCC 7644), Salmonella enteritidis (ATCC 13076), Escherichia coli O157:H7 (NCTC 12900) and Pseudomonas aeruginosa (ATCC 15442), and to identify bioactive functional components including essential oil by GC–MS. Microbial analyses, including disc diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were conducted, whose results were compared with some specific antibiotics. The results of the MIC and MBC analyses indicated that S. enteritidis and L. monocytogenes were more sensitive to the alcoholic extract than P. aeruginosa and E. coli. However, the hydroalcoholic extract showed no MIC and MBC for the tested pathogens. In the disc diffusion method, the alcoholic extract had a great effect on L. monocytogenes. The GC–MS analysis showed phenolic compounds (9.345%), unsaturated and saturated fatty acids (22.901%), alkaloids, and terpenes (58.57%), amino acids (6.279%), squalene (0.233%), and other compounds in the alcoholic extract of Spinach, which has functional roles in biological activities. Furthermore, the most important compounds of the essential oil of the Iranian spinach were durohydroquinone (34.73%), α-terpineol (12.98%), linalool (22.69%), and cineole (13.1%). According to the results, the alcoholic extract and the essential oil of S. oleracea leaves can be a good alternative for antibiotics and can be used in foods and animal feed as a preservative. © 2021 Wiley Periodicals LLC.