Objective To explore the mechanism of resistance-nodulation-cell division (RND) efflux pump genes of multidrug-resistant Acinetobacter baumannii (MDR-AB) AYE strain in influencing biofilm formation. Methods RND efflux pump genes(adeB, adeFGH, adeIJK) of Acinetobacter baumannii AYE strain were knocked out by marker-free gene knockout method, real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to verify the gene knockout of strain efflux pump genes, biofilm formation before and after gene knockout was detected by crystal violet staining, effect of 4 efflux pump inhibitors (PAβN, omeprazole, verapamil and CCCP) on biofilm formation of AYE strain was compared. Results qRT-PCR showed that RND efflux pump genes (adeB, adeFGH, adeIJK genes) of AYE strain were successfully knocked out, AYE△ adeB, AYE△ adeFGH, and AYE△ adeIJK strains were obtained, the amount of biofilm formation was 1.59±0.06, 2.15±0.19 and 1.91±0.02 respectively, the amount of biofilm formation after adeB and adeIJK gene knockout was lower than that before knockout (AYE: 2.31±0.01), difference were all statistically significant (all P < 0.05). Under the effect of PAβN, omeprazole, verapamil and CCCP, the amount of biofilm formation of standard strain AYE of Acinetobacter baumannii were 2.14±0.03, 2.24±0.02, 1.93±0.05 and 2.09±0.04 respectively, which were all lower than those in the control group (AYE: 2.31±0.01), difference was statistically significant (all P < 0.05), PAβN formation was the least. Conclusion Efflux pump genes adeB, adeFGH and adeIJK play important role in the formation of biofilm of MDR-AB AYE, efflux pump inhibitors PAβN, omeprazole, verapamil and CCCP have an inhibitory effect on biofilm formation of Acinetobacter baumannii AYE strain.