Abstract Background Increase in the number of infections caused by Gram-negative bacteria in neutropenic cancer patients has prompted the search for novel therapeutic agents having dual anticancer and antimicrobial properties. Bacteriocins are cationic proteins of prokaryotic origin that have emerged as one of the most promising alternative antimicrobial agents with applications as food preservatives and therapeutic agents. Apart from their antimicrobial activities, bacteriocins are also being explored for their anticancer potential. Results In this study, a broad-spectrum, cell membrane-permeabilizing enterocin with a molecular weight of 65 kDa was purified and characterized from the culture supernatant of vaginal Enterococcus faecium 12a. Enterocin 12a inhibited multidrug-resistant strains of various Gram-negative pathogens such as Salmonella enterica, Shigella flexneri, Vibrio cholerae, Escherichia coli and Gram-positive, Listeria monocytogenes, but had no activities against different strains of gut lactobacilli. The mass spectrometric analysis showed that the enterocin 12a shared partial homology with 4Fe-4S domain-containing redox protein of E. faecalis R712. Further, enterocin 12a selectively inhibited the proliferation of various human cancer cell lines in a dose-dependent manner but not that of normal human peripheral blood mononuclear cells. Enterocin 12a-treated cancer cells showed apoptosis-like morphological changes. Conclusion Enterocin 12a is a novel bacteriocin that has anticancer properties against human cell lines and negligible activity towards non-malignant cells. Therefore, it should be further evaluated for its anticancer potential in animal models.