ABSTRACTAcyl-homoserine lactones (AHLs) are typical quorum-sensing molecules of gram-negative bacteria. Recent evidence suggests that AHLs may also affect gram-positives, although knowledge of these interactions remains scarce. Here, we assessed the effect of AHLs on biofilm formation and transcriptional regulations in the gram-positiveEnterococcus faecalis. FiveE. faecalisstrains were investigated herein. Crystal violet was employed to quantify the biomass formed, and confocal microscopy in combination with SYTO9/PI allowed the visualisation of biofilms’ structure. The differential expression of 10 genes involved in quorum-sensing, biofilm formation and stress responses was evaluated using reverse-transcription-qPCR. The AHL exposure significantly increased biofilm production in strain ATCC 29212 and two isolates from infected dental roots, UmID4 and UmID5. In strains ATCC 29212 and UmID7, AHLs up-regulated the quorum-sensing genes (fsrC,cylA), the adhesinsace,efaAandasa1, together with the glycosyltransferaseepaQ. In strain UmID7, AHL exposure additionally up-regulated two membrane-stress response genes (σV,groEL) associated with increased stress-tolerance and virulence. Altogether, our results demonstrate that AHLs promote biofilm formation and up-regulate a transcriptional network involved in virulence and stress tolerance in severalE. faecalisstrains. These data provide yet-unreported insights intoE. faecalisbiofilm responses to AHLs, a family of molecules long-considered the monopole of gram-negative signalling.