Currently, the majority of antibiotics in clinical use have broad activity spectra, killing pathogenic and beneficial microorganisms indiscriminately. The disruption of the ecological balance of normal flora often results in secondary infections or other antibiotic-associated complications. Therefore, targeted antimicrobial therapies capable of specifically eliminating pathogenic bacteria while retaining the protective benefits of a normal microflora would be advantageous. In this study, we successfully constructed a series of Enterococcus faecalis-targeted antimicrobial peptides from wide-spectrum antimicrobial peptide precursors. These peptides are designed based on fusion of the species-specific peptide pheromone cCF10 and modification of the active region of the antimicrobial peptide. The results showed that cCF10-C4 possessed specific antimicrobial activity against E. faecalis and was not active against other types of bacteria tested. The specificity of this hybrid peptide was shown by the absence of antimicrobial effects in the pheromone-substituted derivative. Further studies indicated that cCF10-C4 and its parent peptide C4 exert their activities by damaging cytoplasmic membrane integrity. The present study reveals the application potential of these molecules as "probiotic" antimicrobials for the control of specific bacterial infections, and it also helps to elucidate the design and construction of species-specific antimicrobials with precise targeting specificity.