The in vivo anti-inflammatory activity of methanolic extract of Prickly pear Opuntia ficus-indica flowers was studied using carrageenan induced rat paw edema model. In order to characterize the extract, the qualitative and quantitative analysis of phenolic compounds of the methanol extract of Opuntia ficus-indica flowers (OFIFE) were performed using LC–MS/MS technique. The main components of OFIFE were phenolic acids and flavonoids. It was observed that the OFIFE inhibited significantly the inflammation as confirmed by the hematological and the histological analysis. The anti-inflammatory effect of OFIFE was associated with the reduction of malonaldialdehyde level (MDA as an index of lipid peroxidation) and an increase in activities of catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH). Overall, the results showed that OFIFE might serve as a natural source for the treatment of inflammation.