Antimicrobial peptides have attracted considerable attention because of their broad-spectrum antimicrobial activity and their low prognostic to induce antibiotic resistance which is the most common source of failure in bacterial infection treatment along with biofilms. The method to design hybrid peptide integrating different functional domains of peptides has many advantages. In this study, we designed an embedded-hybrid peptide R-FV-I16 by replacing a functional defective sequence RR7 with the anti-biofilm sequence FV7 embedded in the middle position of peptide RI16. The results demonstrated that the synthetic hybrid the peptide R-FV-I16 had potent antimicrobial activity over a wide range of Gram-negative and Gram-positive bacteria, as well as anti-biofilm activity. More importantly, R-FV-I16 showed lower hemolytic activity and cytotoxicity. Fluorescent assays demonstrated that R-FV-I16 depolarized the outer and the inner bacterial membranes, while scanning electron microscopy and transmission electron microscopy further indicated that this peptide killed bacterial cells by disrupting the cell membrane, thereby damaging membrane integrity. Results from SEM also provided evidence that R-FV-I16 inherited anti-biofilm activity from the functional peptide sequence FV7. Embedded-hybrid peptides could provide a new pattern for combining different functional domains and showing an effective avenue to screen for novel antimicrobial agents.