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<i>Iphiona mucronata</i> (Family Asteraceae) is widely distributed in the Eastern desert of Egypt. It is a promising plant material for phytochemical analysis and pharmacologic studies, and so far, its specific metabolites and biological activity have not yet been thoroughly investigated. Herein, we report on the detailed phytochemical study using UPLC-Q-TOF-MS approach. This analysis allowed the putative annotation of 48 metabolites belonging to various phytochemical classes, including mostly sesquiterpenes, flavonoids, and phenolic acids. Further, zebrafish embryotoxicity has been carried out, where 100 µg/mL extract incubated for 72 h resulted in a slow touch response of the 10 examined larvae, which might be taken as a sign of a disturbed peripheral nervous system. Results of in vitro testing indicate moderate cytotoxicity towards VERO, FaDu, and HeLa cells with CC₅₀ values between 91.6 and 101.7 µg/mL. However, selective antineoplastic activity in RKO cells with CC₅₀ of 54.5 µg/mL was observed. To the best of our knowledge, this is the first comprehensive profile of <i>I. mucronata</i> secondary metabolites that provides chemical-based evidence for its biological effects. A further investigation should be carried out to precisely define the underlying mechanisms of toxicity.