This study isolated and identified flavonoids from aerial parts of Hyparrhenia hirta Stapf (Poaceae) growing in Tunisia. The material was defatted with petroleum ether and extracted with hot methanol. Ten flavonoids (isoorientin, isoorientin 7-O-rhamnoside, vitexin, isovitexin, tricin 5-O-glucoside, tricin 7-O-glucoside, luteolin 7-O-glucoside, chrysoeriol 7-O-glucoside, luteolin, tricin) were isolated via column chromatography, preparative paper chromatography and semipreparative HPLC, and identified using NMR, UV, acid hydrolysis, LC-MS and comparison with authentic standards. Five additional flavonoids (orientin, vicenin-2, schaftoside, tricin 7-O-rhamnosylglucoside, luteolin 7-O-rhamnosylglucoside) were identified in purified fractions by HPLC-DAD and APCI LC-MS. Chemotaxonomically, the flavonoid profile is typical of Poaceae (containing flavone C-glycosides and tricin 5-O-glucoside), with the exact glycosidic pattern potentially important for classification. Ecologically, the methanol extract stimulated feeding in grass-feeding locusts and Spodoptera frugiperda; isoorientin, tricin and tricin 7-O-glucoside stimulated locust feeding, while luteolin was inactive. The extract also exhibited anti-inflammatory and antioxidant activities, likely due to the flavonoids. Further studies are needed to confirm the compounds responsible for medicinal properties.