A new metabolite, a phenolic derivative, 2-tert-butyl-4-(2-methylhex-5-en-2-yl) phenol (1), together with three known phthalates (2-4), was first isolated from the endophytic fungus Gymnoascus thermotolerans which collected from the roots, stems, and leaves of Euphorbia geniculata for the first time. The structures were determined by mass spectrometric measurements, 1D and 2D NMR investigations, and mass spectroscopic analyses. The antibacterial, antioxidant, and cytotoxic properties of these metabolites were assessed in vitro. 2-tert-butyl-4-(2-methylhex-5-en-2-yl) phenol (1), significantly inhibited the growth of Staphyloccocus aureus and Staphyloccocus epidermidis with minimum inhibitory concentration (MIC) values of 9.0, and 10.5 μM, respectively. In contrast, Escherichia coli, Klebsiella pneumoniae, and Candida albicans showed resistance toward the isolated compounds (1-4),w hich displayed low activity against Candida ciferrii. However, dibutyl phthalate (2) revealed a potent inhibition of spore germination of Fusarium solani with (90.8%) at the highest concentration, 2.5 mg/ml. Moreover, diisooctyl phthalate (3) showed the highest DPPH radical scavenging activity by 72.4% at 500 µg/ml concentration. On the other hand, both dibutyl phthalate (2) and 2-(pentyloxy carbonyl) benzoic acid (4) exhibited potent cytotoxicity against both MDA-MB-231 cancer cells with (IC₅₀ 0.54, 0.64 µM) and SKOV 3 cancer cells with (IC₅₀ 0.75, 0.81 µM), respectively. In addition, the proposed biosynthetic pathways of isolated compounds (1–4), as well as their antimicrobial, antioxidant, and cytotoxicity structure-activity relationship, were discussed.  Our findings provide new insights into the potential utilization of endophytic fungi as innovative reservoirs for the development of antimicrobial and anticancer therapeutic agents.