Objective To explore imipenem-based in vitro and in vivo antibacterial methods against clinically isolated multidrug-resistant Acinetobacter baumannii (MDR-AB). Methods The minimal inhibitory concentrations (MICs) and combined MICs of seven clinically isolated MDR-AB strains against 5 antimicrobial agents (imipenem [IMP], fosfomycin [FOS], amikacin [AMI], tigecycline [TGC], and polymyxin B [PB]) were detected using broth microdilution and checkerboard methods. Fractional inhibitory concentration index (FICI) was calculated. Bactericidal effect of imipenem combined with fosfomycin was determined by time-bactericidal curve. The strains AB6624 and AB0153 were selected to construct the in vitro biofilm model, and biofilm was semi-quantitatively detected by crystal violet staining. Killing effect of imipenem combined with fosfomycin on biofilm bacteria was observed with laser scanning confocal microscopy. Effects of imipenem alone or in combination with fosfomycin on bacterial reactive oxygen species expression were detected by flow cytometry. Wax moth infection model was constructed, and survival rates of wax moth treated with imipenem and fosfomycin alone or in combination were recorded. Results Imipenem combined with fosfomycin had synergistic effect on 5 MDR-AB strains. After combined treatment of imipenem and fosfomycin, colony numbers of AB6624 and AB0153 at 4 and 8 hour were lower than those with single-drug-treatment group by more than 2log₁₀ CFU/mL. The combined use of imipenem and fosfomycin inhibited the formation of AB0153 biofilm and destroyed its structure. It also increased the intracellular ROS level in AB0153, with statistically significant difference (P < 0.05). Compared with control group, imipenem combined with fosfomycin treatment group improved the survival rate of wax moth (median survival time 96 hours), with statistically significant difference (P=0.022). Conclusion Imipenem combined with fosfomycin has synergistic effect on MDR-AB, and may play a role by inhibiting biofilm formation and increasing intracellular ROS level of bacteria.