The inhibitory activity of manuka oil against Herpes simplex virus type 1 (HSV-1) and Herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells (monkey kidney cells) using a plaque reduction assay. In order to determine the mode of antiviral action of the essential oil, manuka oil was added at different times to the cells or viruses during the infection cycle. Both HSV types were significantly inhibited when the viruses were pretreated with manuka oil 1 h prior to cell infection. At non-cytotoxic concentrations of the essential oil, plaque formation was significantly reduced by 99.5 % and 98.9 % for HSV-1 and HSV-2, respectively. The 50 % inhibitory concentration (IC (50)) of manuka oil for virus plaque formation was determined at 0.0001 % v/v ( = 0.96 microg/mL) and 0.00006 % v/v ( = 0.58 microg/mL) for HSV-1 and HSV-2, respectively. On the other hand, pretreatment of host cells with the essential oil before viral infection did not affect plaque formation. After virus penetration into the host cells only replication of HSV-1 particle was significantly inhibited to about 41 % by manuka oil. Flavesone and leptospermone, two characteristic ss-triketones of manuka oil, inhibited the virulence of HSV-1 in the same manner as the essential oil itself. When added at non-cytotoxic concentrations to the virus 1 h prior to cell infection, plaque formation was reduced by 99.1 % and 79.7 % for flavesone and leptospermone, respectively.