Abstract: At present, the development of antibiotic adjuvants to improve the bactericidal effect of traditional antibiotics is one of the effective ways to solve the bacterial resistance. In order to find the new antibiotic adjuvants, the killing tests of Escherichia coli BW25113 and Staphylococcus aureus ATCC25923 were carried out through the exogenous addition of D-trehalose and three major types of bactericidal antibiotics, and the potential bactericidal mechanism of D-trehalose combined with aminoglycoside antibiotics was explored by the exogenous addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the combined group of D-trehalose and aminoglycoside antibiotics. The results showed that D-trehalose combined with aminoglycoside antibiotics had the best killing efficacy on Escherichia coli and Staphylococcus aureus. After the exogenous addition of CCCP in the combined sterilization group, the bacteria could restore the tolerance phenotype. It was indicated that D-trehalose was expected to be used as a new antibiotic adjuvant, suggesting that the antibacterial mechanism of D-trehalose combined with aminoglycoside antibiotics was related to the proton motive force.