Most Arab nations have arid regions where Ephedra transitoria, a plant, flourishes. It has a variety of medical benefits. It belongs to the Ephedraceae family. The plant's aerial portions were procured from the Hail region of Saudi Arabia, methanol was used to extract bioactive phytochemicals with variable solubility in an efficient manner, and the resulting extract was tested on several cancer cell lines. The extract's cytotoxic potential was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay method against standard vinblastine sulfate and for each cell line, the pertinent half maximal inhibitory concentration values were calculated. Additional apoptosis assays were conducted. Also, the antioxidant activity of plant extract was examined using 2, 2-Diphenyl-1-picrylhydrazyl and [2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate)] methods compared to ascorbic acid standard. Also, antimicrobial effect of extract was evaluated on different gram positive and gram-negative bacteria by zone inhibition and minimum inhibitory concentration assay methods. GC-MS analysis was further applied to identify major bioactive substances. Extract showed good cytotoxic activity against both lung and liver cancer cells with relevant half maximal inhibitory concentration values comparable to those of standard. Extract resulted in S-phase arrest, primarily via inducing apoptosis mediated through activating caspase-3, Bax, and p53 proteins. Plant extract showed good antibacterial activities against S. aureus, E. coli and B. subtilis. Also, it showed high antioxidant activity with relevant half maximal inhibitory concentration values comparable to those of standard. Some bioactive compounds were separated and identified by GC-MS analysis such as; ephedrine and quercetin. ephedra transitoria extract possess high cytotoxic activity against lung and liver cancer cells via S-phase arrest mediated by activation of apoptotic proteins. It also possesses high antioxidant and antibacterial activities.