To solve the problem of large-scale growth of wetland reeds, wood vinegar, a by-product of pyrolysed reed wood vinegar, can be used as a natural antimicrobial agent. In this study, we compare the changes in growth and bacterial morphology of Escherichia coli (E. coli) treated with reed wood vinegar at different pyrolysis temperatures (300 °C, 500 °C and 700 °C) and reveal the bacterial inhibition mechanism of reed wood vinegar by RNA-Seq. The results of bacteria inhibitory activity showed that 1/2MIC 500 °C wood vinegar had the most prominent bacteria inhibitory activity. qPCR results showed that reed wood vinegar was able to significantly inhibit the expression of E. coli biofilm and genes related to the cell membrane transporter proteins. Electron microscopy observed that the wood vinegar disrupted the cellular morphology of E. coli, resulting in the crumpling of E. coli cell membranes. RNA-Seq showed the multifaceted antimicrobial effects of wood vinegar and demonstrated that the inhibitory effect of wood vinegar on E. coli was mainly realized through the inhibition of the expression of malE, which is an ATP-binding cassette (ABC) transporter complex of E. coli. In conclusion, our study provides an effective method and a theoretical basis for the mechanism of reed wood vinegar as a natural antimicrobial agent and its pathway of bacterial inhibition.