Abstract: In this work, we adopted β-pinene, which was one of the main components of turpentine oil, to synthesize 36 quaternary ammonium compounds including hydronopyl monoquaternary ammonium salts(1-13), dihydronopyl monoquaternary ammonium salts(14-18) and symmetric dihydronopyl bis-quaternary ammonium salts(19-36). The minimum inhibitory concentrations(MIC) of these compounds against four harmful bacteria such as Escherichia coli, Pseudomonas putida, Staphylococcus aureus and Bacillus subtilis were tested by 96-well microtiter plates, using resazurin as chromogenic agent and kanamycin sulfate as the positive control. The results showed that the antibacterial activities of the synthesized quaternary ammonium compounds were significantly higher than that of the starting material β-pinene and the critical intermediate hydronol. The monoquaternary ammonium salts with hydronopyl and long carbon chain alkyl(10-11), the monoquaternary ammonium salts with dihydronopyl(14-18), and the symmetric dihydronopyl bis-quaternary ammonium salts(19-36) had high antibacterial activity, while the MIC values of most quaternary ammonium compounds were higher than those of kanamycin sulfate(the MIC values against P. putida and the other three bacteria are 10 mg/L and 5 mg/L, respectively). The MIC values of dihydronobuyl dimethyl ammonium bromide(14) against P. putida, S. aureus and B. subtilis were 1.25 mg/L, 2.5 mg/L, 2.5 mg/L, respectively, and the MIC value of dihydronobuyl diethyl ammonium bromide(16) against P. putida was 2.5 mg/L, which was lower than the MIC value of the positive control kanamycin sulfate. Furthermore, the preliminary antibacterial mechanism studies revealed that compound 14 increased the permeability of P. putida cell membranes, resulting in a significant increase in relative conductivity, and at the same time, inhibiting its ATP activity. These hydronopyl quaternary ammonium compounds were expected to be used as potential antimicrobial agents.