Pseudomonas aeruginosa is an opportunistic pathogen responsible for many nosocomial infections. This bacterium uses Quorum Sensing (QS) to generate antimicrobial resistance (AMR) so its disruption is considered a novel approach. The current study describes the antibiofilm and QS inhibitory potential of extract and chemical components from Piper pertomentellum. The methodo- logy included the phytochemical study on the aerial part of the species, the determination of QS inhibition efficacy on Chromobacterium violaceum and the evaluation of the effect on biofilm formation and virulence factors on P. aeruginosa. The phytochemical study led to the isolation and identification of a new piperamide (ethyltembamide 1), together with four known amides (tembamide acetate 2, cepharadione B 3, benzamide 4 and tembamide 5). The results indicated that the ethanolic extract and some fractions reduced violacein production in C. violaceum, however, only the ethanolic extract caused inhibition of biofilm formation of P. aeruginosa on polystyrene microtiter plates. Finally, the investigation determined that molecules (1–5) inhibited the formation of biofilms (50% approximately), while compounds 2–4 can inhibit pyocyanin and elastase production (30–50% approximately). In this way, the study contributes to the determination of the potential of extract and chemical constituents from P pertomentellum to regulate the QS system in P. aeruginosa.