Antibiotic-resistant microorganisms causing a life-threatening infection pose a serious challenge for modern science. The rapidly growing number of incidents for which the use of standard antibiotics is ineffective forces us to develop new alternative methods of killing microorganisms. Antimicrobial proteins and peptides (AMPs) can be promising candidates to solve this problem. Colicin-M is one of the representatives of this group and is naturally produced by Escherichia coli acting on other closely related bacterial strains by disrupting their outer cell membrane. This bacteriocin has huge potential as a potent antimicrobial agent, especially, since it was recognized by the FDA as safe. In this work, we present the expression of colicin M in model transgenic Nicotiana tabacum L. plants. We demonstrate that purified colicin retains its antibacterial activity against the control Escherichia coli strains and clinical isolates of Escherichia coli and Klebsiella pneumoniae. Our results also show that plant-derived ColM is not toxic for L929 and HeLa cell line, which allows us to suppose that plant-based expression could be an alternative production method of such important proteins.