Novel metallic nanoparticles (NPs), such as biosynthesized gold nanoparticles (AuNPs), have been extensively studied because of their wide applications. Consequently, the present investigation aimed at the biosynthesis, characterization, and comparative assessment of biological activities of AuNPs. Biosynthesized AuNPs (ED-AuNPs) were synthesized using aqueous extract of Euphorbia dendroides ( E. dendroides) aerial parts (EDAE), which functions as a reducing and stabilizing agent by its nature. Presence of tannins, saponins, flavonoids, alkaloids, glycosides/or carbohydrates, steroids, and terpenes was revealed by the qualitative phytochemical analysis of EDAE. Characterization of the biosynthesized AuNPs was attained by UV-Visible spectrophotometry, transmission electron microscopy (TEM), XRD, and Fourier transform infrared (FTIR). The cytotoxic effects of ED-AuNPs and EDAE were assessed in vitro against HepG2 and HCT-116 cancer cells. The MTT assay revealed a significant cytotoxic effect of ED-AuNPs and EDAE on HepG2 (IC50 = 41.72 ± 1.26 and 55.26 ± 2.25 μg mL−1) as well as HCT-116 (IC50 = 44.96 ± 3.23 and 69.83 ± 0.96 μg mL−1) cells, thus indicating their potential anticancer activity. Furthermore, ED-AuNPs exhibited potent anti-diabetic activity, with an IC50 value of 19.8 ± 1.97 µg mL−1, pointing to their plausible use as therapeutics in the treatment of diabetes, as well as a promising antimicrobial activity against H. pylori.