HypericumL. (Hypericaceae) extracts have been used for their therapeutic effects; however, not much is known about the immunomodulatory activity of essential oils extracted from this plant. We isolated essential oils from the flowers and leaves ofH. perforatumand analyzed their chemical composition and innate immunomodulatory activity. Analysis of flower (HEOFl) versus leaf (HEOLv) essential oils using gas chromatography-mass spectrometry revealed that HEO(Fl)was comprised mainly of monoterpenes (52.8%), with an abundance of oxygenated monoterpenes, includingcis-p-menth-3-en-1,2-diol (9.1%), alpha-terpineol (6.1%), terpinen-4-ol (7.4%), and limonen-4-ol (3.2%), whereas the sesquiterpenes were found in trace amounts. In contrast, HEO(Lv)was primarily composed of sesquiterpenes (63.2%), including germacrene D (25.7%) and beta-caryophyllene (9.5%). HEO(Lv)also contained oxygenated monoterpenes, including terpinen-4-ol (2.6%), while monoterpene hydrocarbons were found in trace amounts. Both HEO(Fl)and HEO(Lv)inhibited neutrophil Ca(2+)mobilization, chemotaxis, and reactive oxygen species (ROS) production, with HEO(Lv)being much more active than HEOFl. Furthermore, the pure sesquiterpenes germacrene D, beta-caryophyllene, and alpha-humulene also inhibited these neutrophil responses, suggesting that these compounds represented the active components of HEOLv. Although reverse pharmacophore mapping suggested that potential protein targets of germacrene D, beta-caryophyllene, bicyclogermacrene, and alpha-humulene could be PIM1 and mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAK2), a kinase binding affinity assay did not support this finding, implying that other biological targets are involved. Our results provide a cellular and molecular basis to explain at least part of the beneficial immunotherapeutic properties of theH. perforatumessential oils.