en فیتوشیمی Phyochemistry پژوهشي Original Research <strong>Background</strong>: Plantago lanceolata L., a perennial plant of the Plantaginaceae family, is widely distributed across various regions. It is used globally for multiple purposes, including medicinal treatments, food additives, cosmetics, and industrial applications.&nbsp;<br> <strong>Objectives</strong>: This study aimed to investigate the antimicrobial activity and phytochemical profile of root extracts of P. lanceolata.&nbsp;<br> <strong>Methods</strong>: Antibacterial activity was assessed using petroleum ether and aqueous extracts of P. lanceolata roots. The antibacterial activity was assessed using disc diffusion and microtiter broth dilution assays to determine the minimum inhibitory concentration (MIC). Additionally, the minimum bactericidal concentrations (MBCs) were evaluated by culturing the samples on agar media. Gas chromatography-mass spectrometry (GC-MS) was also employed to identify the chemical constituents present in the extracts.&nbsp;<br> <strong>Results</strong>: The petroleum ether extract exhibited the strongest antimicrobial effect, producing an inhibition zone of 15.50 mm against Proteus vulgaris (PTCC 1182). The lowest MIC of 2 mg/mL was observed for both P. vulgaris and Bacillus cereus (ATCC 11778) with this extract, while minimum bactericidal concentration (MBC) values confirmed effectiveness at 3 mg/mL. Chemical analysis revealed that hexadecanoic acid ethyl ester (16.15%) and hexadecanoic acid, methyl ester (5.03%) were the predominant compounds in the petroleum ether extract. In contrast, the aqueous extract contained major artifactual components, such as 3-methoxy-2, 2-dimethyloxirane (30.78%).&nbsp;<br> <strong>Conclusion</strong>: P. lanceolata roots show promise as a natural antibacterial agent with potential applications across pharmaceutical, chemical, food, and medicinal industries. The antibacterial efficacy of the extracts is supported by the identification of active compounds through GC-MS analysis. Disc diffusion method, Gas chromatography-mass spectrometry (GC-MS), Microtiter broth dilution, Minimum inhibitory concentrations (MICs), Plantago lanceolata, Ribwort plantain 37 48 http://pbr.mazums.ac.ir/browse.php?a_code=A-10-1061-6&slc_lang=en&sid=1 Samaneh Rahamouz-Haghighi rahamouz_haghighi.s@yahoo.com 100319475328460012029 100319475328460012029 Yes Agricultural Research, Education and Extension Organization (AREEO), Mashhad, Iran.