Qualitative analysis of the water extract of Mentha x villosa Huds. leaves was performed by liquid chromatography mass spectrometry (LC-MS/MS) and quantitative analysis was made by reverse-phase liquid chromatography coupled with photodiode array detection (LC-DAD). Sixteen phenolic compounds were identified and quantified consisting of 8 phenolic acids/derivatives and 8 flavonoid glycosides (quinic acid, chlorogenic acid, coumaroyl-hexoside, caffeic acid, coumaroylquinic acid, lithospermic acid, rosmarinic acid, salvianolic acid A, luteolin-7-O-glucuronide, luteolin-7-O-glucoside, luteolin-7-O-rutinoside, eriodictyol-7-O-rutinoside, apigenin-7-O-glucuronide, kaempferol-3-O-glucuronide, chrysoeriol-7-O-rutinoside, and hesperetin-7-O-rutinoside). Luteolin-7- O-rutinoside (25.6 ± 0.7 mg/g dry extract) and rosmarinic acid (17.9 ± 0.4 mg/g dry extract) were the most abundant. High antioxidant activity of this phenolic-rich water extract was confirmed in vitro by DPPH and ABTS tests and ex vivo in the ischemia-reperfusion injured rat superior mesenteric artery. Thus, the water extract of M. x villosa leaves seems to be a promising agent in prevention of tissue injury caused by oxidative stress.