The aim of this study was to investigate the phytochemical composition and biological properties of Tunisian propolis from four different regions: Kasserine, Béja, Kèf and Monastir. Ethanolic extracts of propolis were prepared using two extraction methods; solvent and ultrasonic extraction. Total phenolics, flavonoids, ABTS free radical and hydroxyl radicals scavenging abilities, anti-inflammatory, anti-hypertensive, as well as antimicrobial activities of propolis extracts were determined. Identification and quantification of phenolic and flavonoid compounds were performed by using both HPLC-UV and HPLC-ESI-MS. The results revealed high contents of total phenolics and flavonoids and polyphenols extraction was more efficient by sonication. Caffeic acid phenethyl ester (CAPE), galangin, and genistein were the major identified compounds. Antihypertensive activity, evaluated in propolis extracts for first time by HPLC-UV, was higher than 90% for all extracts. Tunisian propolis is an important natural source of polyphenols and flavonoids. The best extraction method was ultrasonic for antioxidants and most of biological activities; conventional method seems to be more suitable for anti-inflammatory activity. Propolis from Béja contains the highest amount of antioxidants and have a stronger potential biological activities. Tunisian propolis could be, therefore, a promising raw material for food and pharmaceutical industry.