LFchimera is a chimerical peptide containing Lactoferricin and Lactoferrampin antimicrobial peptides of bovine lactoferrin, and it has stronger bactericidal activity. Antimicrobial peptides (AMPs) like LFchimera have great potential as an alternative candidate for conventional antibiotics. Plant hairy roots provide suitable platform for fast, easy and cost-effective production of various recombinant proteins. The aim of this study was to express recombinant LFchimera in Nicotiana tabacum hairy roots and investigate its antimicrobial activity. The integration and expression of the transgene in hairy roots were confirmed by PCR and RT-PCR, respectively. LFchimera levels were quantified by ELISA and the presence of LFchimera was verified by SDS-PAGE analysis of root extracts. Biological activity of the plant derived LFchimera was confirmed by investigating the antimicrobial activity of total solution protein against Escherichia coli (ATCC 8739). Hairy root biomass reached to 4.6 g and LFchimera accumulate about 4.8 μg/g fresh weight in Erlenmeyer flasks.