Background and objective: Plantago major belongs to Plantaginaceae family and is an endemic medicinal herb distributed throughout the Iran. Therefor this study is aimed to examine the antimicrobial activity of Plantago major on 4 pathogenic bacteria species including Staphylococcus aureus, Listeria innocua, Escherichia coli and Pseudomonas aeruginosa.Materials and methods: In this study was extracted by maceration method. Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were used as quantitative method, and disk diffusion (Kirby-Baur) and Pure plate methods were applied as qualitative methods.Results: The results of the Kirby-Baur test showed that extracts of Plantago major at the concentration of 20 mg/ml has no inhibitory effect on bacterial growth. The results indicated that the maximum diameter of inhibition zone of aqueous or ethanol extracts of Plantago major in a concentration of 80 mg/ml pertained to the bacterium Staphylococcus aureus. The minimum zone diameter in this concentration was associated with Pseudomonas aeruginosa. The minimum inhibitory concentration of the aqueous extract of Plantago major for Staphylococcus aureus, Listeria innocua, Escherichia coli and Pseudomonas aeruginosa were 25, 50, 100 and 200 mg/ml, respectively, and the MIC of ethanol extract were 12.5, 25, 50 and 100 mg/ml, respectively. The minimum bactericidal concentration of the aqueous extract of Plantago major for Staphylococcus aureus, Listeria innocua, Escherichia coli and Pseudomonas aeruginosa were 50, 50, 200 and 400 mg/ml, respectively, and the MIC of ethanol extract were 25, 25, 50 and 200 mg/ml, respectively.Conclusion: The ethanolic extract of the Plantago major compared with the Vancomycin antibiotic had more inhibitory effect on studied bacteria. Furthermore, Plantago major extracts showed greater inhibitory effect on Gram-positive bacteria in comparison with Gram-negative bacteria.