The presence of Pseudomonas aeruginosa in cutaneous wounds is of clinical significance and can lead to persistent infections. Manuka honey has gained ground in clinical settings due to its effective therapeutic action and broad spectrum of antibacterial activity. In this study, the effect of manuka honey on P. aeruginosa was investigated using MIC, MBC, growth kinetics, confocal microscopy, atomic force microscopy and real-time PCR. A bactericidal mode of action for manuka honey against P. aeruginosa was deduced (12 %, w/v, MIC; 16 %, w/v, MBC) and confirmed by confocal and atomic force microscopy, which showed extensive cell lysis after 60 min exposure to inhibitory concentrations of manuka honey. The inability of honey-treated cells to form microcolonies was demonstrated and investigated using Q-PCR for three key microcolony-forming genes: algD, lasR and oprF. The expression of algD increased 16-fold whereas oprF expression decreased 10-fold following honey treatment; lasR expression remained unaltered. These findings confirm that manuka honey is effective at inducing cell lysis and identify two targets, at the genetic level, that might be involved in this process.