<p>The advent and global spread of plasmid-carried resistance genes, including the tigecycline resistance gene <i>tet</i>(X3/X4) and the colistin resistance gene <i>mcr-1</i>, present a substantial threat to public health that requires immediate resolution. This study successfully screened and identified commercial anti-parasitic drugs that efficiently inhibit Tet(X4) enzyme activity (IC₅₀ = 79.42&#xa0;µg/mL), revealing that mefloquine exhibited a synergistic antibacterial effect with tetracyclines against <i>tet</i>(X3/X4)-positive bacteria (FIC &lt; 0.5) and an additive effect with tetracyclines against <i>tet</i>(X3/X4)-negative bacteria (0.5 &lt; FIC &lt; 1, excluding minocycline). Molecular dynamics simulations and checkerboard minimum inhibitory concentration (MIC) tests on Tet(X4) mutants in amino acid residues demonstrated that mefloquine’s interaction with the active site of Tet(X4) reduces its activity. The energy contribution of the binding site residues involves PHE221, PHE316, GLY233, PRO315, PHE232, MET212, ALA222 and HIE231, which establish robust interactions with mefloquine (Δ_{<i>Etotal</i>} of ≤ − 0.5&#xa0;kcal/mol). Moreover, findings from metabonomics, bacterial membrane analysis, ROS detection, NAD⁺/NADH ratio determination, and intracellular ATP determination indicated that mefloquine may significantly influence bacterial energy metabolism (<i>P</i> &lt; 0.05). Furthermore, in the mouse infection model of <i>E. coli</i> 47EC, the combination of methacycline and mefloquine, demonstrated significant therapeutic advantages, as evidenced by an increased survival rate (from 20.00% to 80.00%), decreased sepsis score, and decreased bacterial load. Our work is the first to reveal that the commercial medicine mefloquine can prolong the efficacy of tetracycline and treat infections caused by drug-resistant <i>Enterobacteriaceae</i> in clinical environments.</p> Graphical Abstract <p></p>