Today Antibiotic resistance is one of the global concerns in medicine. Due to the increasing emergence of antibiotic-resistant bacteria and the problems with their treatment, the use of alternative methods to increase effectiveness and reduce drug resistance is important. In traditional medicine, the use of ferula plant due to its various active chemical compounds with antibacterial, antioxidant and cytotoxic activity, has attracted the attention of researchers. Because, many studies have been performed on other species of Ferula, the aim of this study was to investigate the antimicrobial effect of Ferula macrocolea extract on 4 standard strains of gram-positive and gram-negative bacteria. The present study was performed on 4 standard bacterial strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Bacillus subtilis. First, Ferula essential oil was extracted. Microbial suspension with specific and standard concentrations was prepared in Müller-Hinton Broth medium. Antimicrobial properties of the essential oil on the desired bacteria were performed using two methods of disk diffusion and microbroth dilution according to the CLSI standards. The results of this study showed that Ferula extract has different antimicrobial effect on all strains. The highest and lowest antimicrobial and inhibitory effects of this extract were reported on B. subtilis and S. epidermidis, respectively. Examination of the antibacterial effect of essential oil from F. macrocolea showed that this extract has different lethal and inhibitory effects on gram-positive and gram-negative bacteria.