S. aureus under the biofilm mode of growth is often related to several nosocomial infections, more frequently associated with indwelling medical devices (catheters, prostheses, portacaths or heart valves). As a biofilm, the biopolymer matrix provides an excellent growth medium, increasing the tolerance to antibiotics and host immune system. To date, the antimicrobial therapy alone is not effective. A novel strategy to prevent biofilm formation is based on the interference with the bacterial cell–cell communication, a process known as quorum sensing (QS) and mediated by the RNA-III-activating peptide (RAP) and its target protein TRAP (Target of RAP). The RNAIII inhibiting peptide (RIP) is able to inhibit S. aureus pathogenesis by disrupting QS mechanism competing with RAP, thus inhibiting the phosphorylation of TRAP. This alteration leads to a reduced adhesion and to the inhibition of RNAIII synthesis, with the subsequent suppression of toxins synthesis. The present paper will provide an overview on the activity and potential applications of RIP as biofilm inhibiting compound, useful in the management of S. aureus biofilm infections. Moreover, medicinal chemistry strategies have been examined to better understand which modifications and/or structure alterations were able to produce new derivatives of this QS inhibitor with an improved antibiofilm activity.