The antimicrobial quantitative structure–activity relationship of plant flavonoids against Gram-positive bacteria was established in our previous works, and the cell membrane was confirmed as a major site of action. To investigate whether plant flavonoids have similar antibacterial effects and mechanisms against both Gram-negative and Gram-positive bacteria, here, the minimum inhibitory concentrations (MICs) of 37 plant flavonoids against Escherichia coli were determined using the microdilution broth method, and then the correlation between their lipophilic parameter ACD/LogP or LogD_7.40 value and their MIC was analyzed. Simultaneously, the correlation between the ACD/LogP or LogD_7.40 value and the MIC of 46 plant flavonoids reported in literature against E. coli was also analyzed. Both sets of results showed that there is a significant correlation between the LogP value and the MIC of plant flavonoids against Gram-negative bacteria. However, it is difficult to effectively predict the MIC of plant flavonoids against Gram-negative bacteria from their lipophilic parameters. By comparing two regression curves derived from plant flavonoids against Gram-negative and Gram-positive bacteria, it was further discovered that the antibacterial activities of most plant flavonoids against Gram-negative bacteria are stronger than those against Gram-positive bacteria when their LogP values are less than about 3.0, but the opposite is true when their LogP values are more than about 3.6. Moreover, this comparison also suggests that unlike mainly acting on the cell membrane of Gram-positive bacteria, plant flavonoids have multiple mechanisms against Gram-negative species, while the cell membrane is also an important action site among them. Combined with the correlation analyses between the enzyme inhibitory activity and the LogP value of the reported flavonoids, it was further suggested that DNA gyrase is another important target of plant flavonoids against Gram-negative bacteria.