Urospermum picroides is a medicinal plant was founded in North coast, Egypt. Endophytic fungi of this medicinal plant in Egypt is poorly known and thus, this study aimed towards estimation of the biological activities of endophytic fungi from Egyptian U.picroides. Fusarium oxysporum and Phoma herbarum were isolated from U.picroides and identified based on phylogenetic analysis. Ethyl acetate extracts of F.oxysporum and P.herbarum gave high antioxidant activities (26.2 and 51.7%, respectively). Moreover, they showed highly antitumor activities with some significant morphological changes of characteristic apoptosis accompanied by up regulation of both p53 and Bax for F.oxysporum and P.herbarum extracts (1.55, 2.14 for p53 and 1.24, 2.2 for Bax, respectively) with down regulation of Bcl-2 (0.18 and 0.16, respectively). Ethyl acetate extract of F.oxysporum (EAFE) showed greatest antimicrobial activity against Trichophyton mentagrophytes (70 mm), while ethyl acetate extract of P.herbarum (EAPE) showed highest antimicrobial activity against Bacillus cereus (37 mm) and Candida albicans (35 mm). Scanning electron microscope micrographs showed major abnormalities for tested microorganisms after treatment with (EAFE and EAPE) resulting in complete alternation in their morphology. GC-MS results showed 30 biologically active compounds for both extracts. The most significant in EAFE was Diisooctyl phthalate with relative levels (74.9%), while Benzoic acid, 3, 5-bis (1, 1-dimethylethyl)-4-hydroxy-was the most active compound in EAPE (61.7%). These results proposed that ethyl acetate extracts of endophytic fungi from the Egyptian U.picroides showed to be promising novel as antioxidant, antitumor agents and antimicrobial with further phytochemical studies.