The effects of the structures of non-ionic surfactants on the uptake of butyl p-hydroxybenzoate (BP) into Escherichia coli cells and its antibacterial activity were systematically studied using polyoxyethylene alkyl ethers (PAEs) possessing various oxyethylene and hydrocarbon chain lengths. The uptake of BP into bacterial cells in an aqueous PAE solution was proportional to free BP in an aqueous phase, depending on the structures of PAEs. The antibacterial activity of BP decreased in the presence of PAEs, whereas it was greater than that anticipated from free BP. However, only PAE with 12 carbons in the hydrophobic group caused unusual increases in the uptake and antibacterial activity of BP, and the surfactant was more extensively incorporated into bacterial cells, differing from other PAEs, which were much less incorporated. The PAEs were thus concluded to increase the susceptibility of bacteria against BP due to direct interactions with the cells. Particularly, the PAE with 12 carbons in the hydrophobic group, which penetrated abundantly into the cells, might result in an increase in the fluidity of the cellular lipid matrix and a decrease in the resistance of drug permeation.