Origanum minutiflorum, an endemic species in Turkiye, is used for both food and medicinal purposes. 70% ethanol extract of aerial parts of O. minutiflorum was subjected to liquid-liquid extraction using; n-hexane, dichloromethane, ethyl acetate and n-butanol. The inhibitory activity of these fractions on lipoxygenase (LOX), cyclooxygenase (COX) and matrix metalloproteinase-9 (MMP-9) was investigated at 100 µg/mL concentration. The LOX enzyme was most effectively inhibited by the dichloromethane fraction (82.33%), whereas the COX-1, COX-2 and MMP-9 enzymes were most effectively inhibited by the ethyl acetate fraction (78.50%, 83.96% and 45.11%, respectively). The ethyl acetate fraction was selected for activity-guided fractionation based on the COX enzymes’ inhibitory activities by column chromatography. Only the ethyl acetate sub-fraction-1 showed inhibition on the COX-1 with 65.78%. The COX-2 inhibitory activities of the sub-fractions ranged from 59.96% to 97.62%. The main components were determined by HPLC-MS/MS as jaceidine in the dichloromethane fraction and rosmarinic acid in the ethyl acetate fraction. As far as we know, this study is the first to correlate the anti-inflammatory activity of O. minutiflorum extracts with LOX, COX and MMP-9 enzyme pathways and their secondary metabolites. These data showed that O. minutiflorum may contribute to other studies on natural origin anti-inflammatory agents.