The polyphenolic-rich extract of Tunisian Origanum glandulosum Desf. was investigated for its phenolic composition, antioxidant activity and potential to contribute to the dietary management of type II diabetes. By mean of an ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-PDA-ESI-MS) 16 phenolics were characterized. The major detected compounds are caffeic acid, luteloin glucoside, xanthomicrol and carvacrol. The polyphenolic-rich extract presented a notable antioxidant activity similar to that of the synthetic antioxidant Trolox. It was found to inhibit, in a concentration dependent manner, the alpha-amylase activity with an IC50 value (8.22 mg/mL) comparable to that of Acarbose (6.12 mg/mL). Overall, our findings suggest that O. glandulosum could be considered as a valuable source of high-value phytochemicals with pharmaceutical and functional food additive applications.