Many Gram-negative bacteria produceacyl-homoserine lactones (AHLs), quorum sensing (QS) molecules that can be enzymatically inactivated by quorum quenching (QQ) processes; this approach is considered an emerging antimicrobial alternative. In this study, kinetic parameters of several AHLs hydrolyzed by penicillin acylase from ( PA) and aculeacin A acylase from ( AAC) have been determined. Both enzymes catalyze efficiently the amide bond hydrolysis in AHLs with different acyl chain moieties (with or without 3-oxo modification) and exhibit a clear preference for AHLs with long acyl chains (C12-HSL > C14-HSL > C10-HSL > C8-HSL for PA, whereas C14-HSL > C12-HSL > C10-HSL > C8-HSL for AAC). Involvement of PA and AAC in QQ processes was demonstrated by CV026-based bioassays and inhibition of biofilm formation by , a process controlled by QS molecules, suggesting the application of these multifunctional enzymes as quorum quenching agents, this being the first time that quorum quenching activity was shown by an aculeacin A acylase. In addition, a phylogenetic study suggests that PA and AAC could be part of a new family of actinomycete acylases, with a preference for substrates with long aliphatic acyl chains, and likely involved in QQ processes.