ABSTRACTIn relation to emerging multiresistant bacteria, development of antimicrobials and new treatment strategies of infections should be expected to become a high-priority research area. Quorum sensing (QS), a communication system used by pathogenic bacteria likePseudomonas aeruginosato synchronize the expression of specific genes involved in pathogenicity, is a possible drug target. Previousin vitroandin vivostudies revealed a significant inhibition ofP. aeruginosaQS by crude garlic extract. By bioassay-guided fractionation of garlic extracts, we determined the primary QS inhibitor present in garlic to be ajoene, a sulfur-containing compound with potential as an antipathogenic drug. By comprehensivein vitroandin vivostudies, the effect of synthetic ajoene towardP. aeruginosawas elucidated. DNA microarray studies of ajoene-treatedP. aeruginosacultures revealed a concentration-dependent attenuation of a few but central QS-controlled virulence factors, including rhamnolipid. Furthermore, ajoene treatment ofin vitrobiofilms demonstrated a clear synergistic, antimicrobial effect with tobramycin on biofilm killing and a cease in lytic necrosis of polymorphonuclear leukocytes. Furthermore, in a mouse model of pulmonary infection, a significant clearing of infectingP. aeruginosawas detected in ajoene-treated mice compared to a nontreated control group. This study adds to the list of examples demonstrating the potential of QS-interfering compounds in the treatment of bacterial infections.