Enterococci are the leading cause of nosocomial infections worldwide and acquired resistance to a variety of antibiotics. Antimicrobial peptides represent a promising molecule against the antibiotic resistance in bacteria and an indispensable component of the innate immune system. The aim of the study was to develop an antimicrobial peptide against vancomycin-resistant enterococci (VRE). We have designed a series of peptides based on Sapecin B as template. An in vitro antibacterial study of synthetic peptide BF2 against the clinical isolates of vancomycin-resistant and control strains of enterococci showed rapid killing effect on enterococci by killing 99.9% of bacterial cells in 60 min and susceptibility at minimum inhibitory concentration (MIC) range of 6.25-12.5 μg/mL. Synergy of BF2 was observed in combination with vancomycin and teicoplanin. The peptide was bactericidal and nontoxic to mammalian cells. An in vivo study also revealed the antibacterial activity against enterococci-infected Wistar albino rats. BF2 may be used synergistically with antibiotics.