Abstract Due to the emergence of multidrug-resistant bacteria, treatment options for infectious diseases are decreasing. Bacteriocins are small antimicrobial peptides produced by numerous bacteria that offer alternative therapeutic strategies to combat multidrug-resistant bacterial infections. We evaluated the cloning, functional expression, and antimicrobial activities of enterocin P (EntP), a class II bacteriocin member, in Chinese hamster ovary (CHO) cells. A synthetic gene matching CHO cell codon usage was designed from the known mature amino acid sequence of EntP and cloned into the protein expression vector pcDNA™3.1(+). CHO cells were transformed with the recombinant plasmid and cultured, and the recombinant protein was purified by affinity chromatography. Antimicrobial activities of the recombinant EntP were evaluated on Gram-positive, Gram-negative, and multidrug-resistant pathogens. Recombinant EntP inhibited growth of a variety of bacteria, including pathogenic species known to cause nosocomial infections, often with multidrug-resistant strains. In addition, recombinant EntP demonstrated broad antimicrobial activities in both high salt medium and human plasma and was stable at high temperatures. The broad antimicrobial activity and stability of EntP make it an attractive therapeutic candidate, particularly for treatment of multidrug-resistant bacterial infections.