Staphylococcal bacteria are a prevalent cause of infections associated with foreign bodies and indwelling medical devices. Bacteria are capable of escaping antibiotic treatment through encapsulation into biofilms. RNA III-inhibiting peptide (RIP) is a heptapeptide that inhibits staphylococcal biofilm formation by obstructing quorum-sensing mechanisms. K(4)-S4(1-13)(a) is a 13-residue dermaseptin derivative (DD(13)) believed to kill bacteria via membrane disruption. We tested each of these peptides as well as a hybrid construct, DD(13)-RIP, for their ability to inhibit bacterial proliferation and suppress quorum sensing in vitro and for their efficacy in preventing staphylococcal infection in a rat graft infection model with methicillin-resistant Staphylococcus aureus (MRSA) or S. epidermidis (MRSE). In vitro, proliferation assays demonstrated that RIP had no inhibitory effect, while DD(13)-RIP and DD(13) were equally effective, and that the chimeric peptide but not DD(13) was slightly more effective than RIP in inhibiting RNA III synthesis, a regulatory RNA molecule important for staphylococcal pathogenesis. In vivo, the three peptides reduced graft-associated bacterial load in a dose-dependent manner, but the hybrid peptide was most potent in totally preventing staphylococcal infections at the lowest dose. In addition, each of the peptides acted synergistically with antibiotics. The data indicate that RIP and DD(13) act in synergy by attacking bacteria simultaneously by two different mechanisms. Such a chimeric peptide may be useful for coating medical devices to prevent drug-resistant staphylococcal infections.