Natural berberine-derived azolyl ethanols as new structural antibacterial agents were designed and synthesized for fighting with dreadful bacterial resistance. Partial target molecules exhibited potent activity against the tested strains, particularly, nitroimidazole derivative <b>4d</b> and benzothiazole-2-thoil compound <b>18b</b>, with low cytotoxicity both exerted strong antibacterial activities against multidrug-resistant <i>Escherichia coli</i> at low concentrations as 0.007 and 0.006 mM, respectively. Meanwhile, the active compounds <b>4d</b> and <b>18b</b> possessed the ability to rapidly kill bacteria and observably eradicate the <i>E. coli</i> biofilm by reducing exopolysaccharide content to prevent bacterial adhesion, which was conducive to alleviating the development of <i>E. coli</i> resistance. Preliminary mechanistic explorations suggested that the excellent antibacterial potential of molecules <b>4d</b> and <b>18b</b> might be attributed to their ability to disintegrate membrane, accelerate ROS accumulation, reduce bacterial metabolism, and intercalate into DNA groove. These results provided powerful information for the further exploitation of natural berberine derivatives against bacterial pathogens.