In 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 like Pseudomonas aeruginosa to synchronise the expression of specific genes involved in pathogenicity, is a possible drug target. Previous in vitro and in vivo studies revealed a significant inhibition of P. aeruginosa QS by crude garlic extract. By bioassay-guided fractionation of garlic extracts we determined the primary QS inhibitor present in garlic as ajoene, a sulfur-containing compound with potential as an antipathogenic drug. By comprehensive in vitro and in vivo studies of the effect of synthetic ajoene towards P. aeruginosa was elucidated. DNA microarray studies of ajoene treated P. aeruginosa cultures revealed a concentration dependent attenuation of a few, but central QS controlled virulence factors including rhamnolipid. Furthermore, ajoene treatment of in vitro biofilms demonstrated a clear synergistic, antimicrobial effect with tobramycin on biofilm killing and a cease in lytic necrosis of polymorphonuclear leukocytes. Furthermore, in a pulmonary infectious mouse model a significant clearing of infecting P. aeruginosa was detected in ajoene-treated mice compared to a non-treated control group. This study adds to the list of examples demonstrating the potential of QS interfering compounds in the treatment of bacterial infections.