Objectives: The present study aimed to investigate and isolate different phytochemical constituents and assess the antioxidant and antimicrobial activities of the total methanol extract and different fractions of Phagnalon barbeyanum aerial parts. Methods: Phytochemical screening of Phagnalon barbeyanum aerial parts was performed using specific test for each class of compounds. Different chromatographic techniques were used to isolate and purify compounds and their structures were elucidated by using different spectral techniques (¹H NMR and ¹³C NMR). The antioxidant activity was evaluated by DPPH radical scavenging assay and antimicrobial activity was done by standard agar well diffusion assay. Results: Phytochemical investigation of Phagnalon barbeyanum aerial partswas done forthe presence ofcarbohydrates and or glycosides, sterols and or triterpene, flavonoids, tannins and saponins and revealed the absence of alkaloids and anthraquinones. Compounds; β-sitosterol (1), apigenin (2) and β-sitosterol-3-O-β-D- glucopyranoside (3) were isolated from methylene chloride and ethyl acetate fractions. The ethyl acetate fraction showed highest antioxidant activity followed by n-butanol and methylene chloride fractions. The total methanol extract and n-hexane fractions showed lowest antioxidant activity. The ethyl acetate fraction and total methanol extract showed moderate activity against Staphylococcus aureus and Vancomycin resistant Staphylococcus aureus. Conclusion: The aerial parts of Phagnalon barbeyanum is a rich source of different classes of active constituents as phenolics. The total methanol extract of Phagnalon barbeyanum aerial parts and some of its fractionated concentrates could be considered as antioxidant and antimicrobial agents.