Engineering of efflux pumps is a promising way to improve host's tolerance to biofuels such as medium-chain alcohols (CmOHs); however, this strategy is restricted by poor understanding of the efflux pumps engaged in extrusion of solvents. In this study, several Escherichia coli mutants of multidrug transporters were evaluated for isoprenol tolerance. Susceptible phenotypes were observed in the mutants with individual deletion of six transporters, AcrD, EmrAB, MacAB, MdtBC, MdtJI and YdiM, whereas inactivation of AcrAB transporter resulted in an improved tolerance to isoprenol and other CmOHs. AcrAB is the major transporter forming tripartite transperiplasmic complex with outer membrane channel TolC for direct extrusion of toxic molecules in E. coli. The AcrAB inactivation enables to enhance TolC availability for the multidrug transporters associated with extrusion of CmOHs and increase the tolerance to CmOHs including isoprenol. It is assumed that outer membrane channel TolC plays an important role in extrusion of isoprenol and other CmOHs.