Objective To investigate the antimicrobial activity of the bacteriocin (Enterocin-162) secreted by Enterococcus faecalis SHAMU-EF162 strain against clinically common multidrug-resistant organisms (MDROs). Methods The growth kinetics curve of the SHAMU-EF162 strain was monitored through the microcultivation assay. The antagonistic activity of the SHAMU-EF162 strain against Staphylococcus aureus (S. aureus) was eva-luated using Oxford cup method. Enterocin-162 crude extract was prepared by macroporous resin adsorption, gradient ethanol elution, rotary evaporation drying, and reconstitution. The protein content in the crude extract was detected by Coomassie brilliant blue staining. The thermal, pH, and protease stability of Enterocin-162, as well as its antimicrobial activity against methicillin-resistant S. aureus (MRSA), multidrug-resistant Pseudomonas aeruginosa (MDR-PA), extended-spectrum β-lactamases-producing Escherichia coli (ESBLs-EC), and carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolates were assessed by Oxford cup method. Results Enterocin-162 can withstand exposure to 100℃ heat, pH 2.0, pH 12.0, chymotrypsin, bromelain, trypsin, proteinase K, and the mixture of four proteases. The antimicrobial activity of 30 μL crude extract of Enterocin-162 (containing approximately 14.8 μg of protein or polypeptide)against clinical isolates of MRSA, MDR-PA, ESBLs-EC, and CRKP was superior to those of antimicrobial disks of vancomycin, polymyxin, epicycline, amikacin, and levofloxacin. Conclusion Enterocin-162 is likely to be a stable, efficient, and broad-spectrum new class I bacteriocin, with the potential to be developed into a novel antimicrobial agent, and providing a new option for the treatment of common MDRO infection in clinical practice.