Hyssopus cuspidatus is a famous spice and an aromatic vegetable. Few information could be available concerning its non-volatile chemical composition and bioactivities. Preliminary bioactive evaluations on the crude ethanol extract and its four fractions disclosed that the ethyl acetate fraction (EAF) exhibited antioxidant and antimicrobial bioactivities. LC-MS/MS analysis of EAF helped to identify sixty-four compounds, and phenolic compounds were the dominant components. Systematic separation and purification of EAF led to the isolation of thirty-four compounds. Six compounds were identified to be new and eighteen compounds were discovered from H. cuspidatus for the first time. Rosmarinic acid, methyl rosmarinate, butyl rosmarinate and salvigenin were the major components of EAF and their contents were determined. Most of isolated compounds exhibited significant or moderate antioxidant and antimicrobial activities. This research supported the edible application of H. cuspidatus and disclosed the potency of it as a natural antioxidant and antimicrobial food additive.