© 2019 Wiley Periodicals, Inc.The phenolic contents and antioxidant, anticancer, antidiabetic, and anticholinergic potentials of four endemic Gysophila taxa (G. pallida, G. arrosti, G. tuberculosa, and G. eriocalyx) were investigated. The HPLC analysis showed that methanol extracts of all the tested species were richer in phenolics than water extracts. 3,4-dihydroxybenzoic acid, p-hydroxybenzoic acid, vanillin, syringic acid, and p-coumaric acid were detected in all extracts. In parallel to the phenolic contents, methanol extracts displayed comparatively higher antioxidant activity than water extracts. Additionally, all extracts exhibited dose-dependent antiproliferative activity on the cancer cell lines with lower IC50 values changing from 0.170 to 1.805 mg/ml. Moreover, the extracts impressively inhibited the acetylcholinesterase (0.63–26.04), butyrylcholinesterase (3.66–10.73), and α-glycosidase (98.52–235.55) enzymes with very low IC50 (mg/ml) values. Together, the present results indicate that Gysophila taxa have various biological activities together with higher phenolic contents. Hence, these species hold good potential for use in the pharmaceutical industry. Practical applications: Gypsophila taxa having numerous biological activities have been used for different purpose in folk medicine as well as their use in the food industry. The obtained results of the current study indicated that the extracts of Gypsophila taxa are rich in phenolics and flavonoids with powerful antioxidant and antiproliferative activity against different type of cancer cell lines. In addition, the extracts obtained from these taxa showed notable antidiabetic and anticholinergics effects. Gypsophila taxa could be used as a natural material to develop anticancer, antidiabetic, and anticholinergic drugs.