Gram-negative bacteria rely on pili assembly for pathogenicity, with the chaperone-usher (CU) pathway regulating pilus biogenesis. Nitazoxanide (NTZ) inhibits CU pathway-mediated P pilus biogenesis by specifically interfering with the proper folding of the outer membrane protein (OMP) usher, primarily mediated by the β-barrel assembly machinery (BAM) complex. In this study, we identified the BAM complex components BamB and the BamA POTRA2 domain as key binding targets for NTZ. Molecular dynamics simulations and Bio-Layer Interferometry revealed that BamB residues S61 and R195 are critical for NTZ binding. NTZ activated the Cpx two-component system and induced inner membrane perturbations, which resulted from the accumulation of misfolded P pilus subunits. Upregulation of the ibpAB gene, which protects the bacteria against NTZ-induced oxidative stress, was also observed. Importantly, NTZ combined with polymyxin B enhanced the latter's antibacterial activity against both susceptible and MCR-positive E. coli strains. This enhancement was achieved through NTZ-induced increases in inner membrane permeability, oxidative stress, and inhibition of efflux pump activity and biofilm formation. This study provides new insights into the antimicrobial mechanism of NTZ and highlights its potential as an antibiotic adjuvant by targeting BamB to inhibit the CU pathway, restoring the efficacy of polymyxin B against multidrug-resistant bacteria.