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Antibiotics can be classified based upon drug-target interaction and lethality. Bactericidal drugs, which target cell wall/membrane synthesis, DNA replication and repair or protein synthesis, induce hydroxyl radical leading to the bacterial cell death with extreme efficacy. The antibiotic mechanism of the bactericidal drugs is followed by stimulating oxidation of nicotinamide adenine dinucleotide hydrate (NADH) via the electron transport chain that is dependent upon the tricarboxylic acid (TCA) cycle. The five herbal samples, Houttuynia cordata Thunb (HCT), Chrysanthemum lavandulifolum (CL), Patrinia scabiosaefolia (PS), Angelica dahurica Bentham et Hooker (ADBH) and Agrimonia pilosa (AP) were extracted to acquire the fractions, and then inoculated to investigate their antibiotic effect against the bacteria Escherichia coli O157:H7 through the disk diffusion method. Total RNA of the treated cells was isolated to compare and analyze their gene expression profiling. Results from this study indicate that the molecular mechanisms of simultaneous multi-target antibiotic efficacy of the five herbal samples include bacterial cell wall/membrane synthesis, DNA replication and repair, and protein synthesis. The antibiotic mechanisms were associated with the hydroxyl radical damage followed by overproduction of superoxide. Moreover, inhibition of multidrug resistance system and etiological factors are encouraging development of novel antibiotics. Our study suggests that the five herbal samples have a molecular mechanism similar to that of bactericidal drugs that is induction of hydroxyl radical damage which leads to bacterial cell death.