Objective To explore the activity and mechanism of polymyxin B combined with novel β-lactam/β-lactamase inhibitor combinations on biofilm of polymyxin B-resistant Pseudomonas aeruginosa and Klebsiella pneumo-niae. Methods The minimum inhibitory concentration (MIC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC) of all antimicrobial agents were determined by micro-broth dilution method and MBEC^TM assay. The crystal violet staining method was adopted to evaluate the effect of polymyxin B combined with cefepime/avibactam, ceftazidime/avibactam, meropenem/avibactam, aztreonam/avibactam, meropenem/vaborbactam, and imipenem/relebactam at sub-MIC doses on inhibition of biofilm formation and eradication of mature biofilm. The best combination scheme for anti-biofilm activity was screened out, and anti-biofilm mechanism of this combination scheme was preliminarily explored with phenol-sulfuric acid method, bacterial motility test, and quorum sensing inhibition test. Results The MBIC and MBEC of all antimicrobial agents were higher than MIC. The combination regimen based on polymyxin B could inhibit the formation of biofilms and eradicate mature biofilm in Pseudomonas aeruginosa and Klebsiella pneumoniae. The combination of polymyxin B with cefepime/avibactam had the highest inhibition and eradication rates, ranging 67.99%-90.16% and 58.26%-63.86%, respectively. The combination of polymyxin B and cefepime/avibactam could inhibit the extracellular polysaccharides of Klebsiella pneumoniae, with inhibition rates of 34.04%-61.10%, this combination could also reduce the swimming and twitching motility diameters of bacteria. Cefepime/avibactam monotherapy on quorum sen-sing signaling molecules presented concentration dependent inhibitory effect, and when combined with polymyxin B, the inhibitory effect was consistent with that of monotherapy. Conclusion Polymyxin B and cefepime/avibactam may be potential scheme for clinical treatment for severe biofilm-associated infection caused by polymyxin B-resistant strains, and their mechanisms may be related to the inhibition of bacterial extracellular polysaccharides and motility.