We have recently found that 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone [TF18] exhibited the most potent antibacterial activity among 30 trifluoromethyl ketones against various prokaryotes, such as Escherichia coli (E. coli). In the present study, the inhibition of E. coli motility by TF18 was investigated. TF18 showed the lowest minimum inhibitory concentration (MIC) and highest inhibitory effect on the motility of E. coli strains. The wild-type E. coli was more sensitive to inhibition of motility than its proton pump-deleted mutant strain at subinhibitory concentrations. These data suggest that one of the targets of the antibacterial effect of the trifluoromethyl ketone is the proton pump system.