The Genus Cleome has many beneficial properties and phytochemical components, and considerable potential as a novel therapeutic source. This study assessed the phytochemical composition, antiviral activity against HAV, anticancer effects against HepG2 (hepatocellular carcinoma cell line) and MCF7 (human cancer breast cell line), and antimicrobial efficacy of an ethanol 70% aerial parts extract from Cleome droserifolia in vitro. In 70% ethanol extract of Cleome droserifolia, flavonoids, tannins, alkaloids, saponins, terpenes, and sterols were identified. The Cleome droserifolia extract showed great efficacy against HAV, the HepG2 and MCF7 cell lines, with a selective index of 3.85, 2.253 and 1.18 respectively. It is also effective against Gram-positive Bacillus cereus and Gram-negative Enterobacter cloacae bacteria and Fungus; Candida albicans. Benzene-1-pentyloctyl (5.77%), Benzene-1-butylheptyl (5.06%), Benzene-1-methyldecyl (4.93%), Benzene-1-methyldecyl (4.77%), Benzene-1-methylundecyl (4.66%) and Benzene-1-pentylheptyl (4.38%) were found to be the most abundant components from Cleome droserifolia petroleum ether extract by using a Gas Chromatography–Mass Spectrum (GC-MS) analysis. Conversely, the results for indolizine, retinal, and retinol were 0.44%, 0.54%, and 0.06%, respectively.