Background: Dipterygium glaucum Decne. herb is one of the common traditional plants with multiple medicinal uses. Objective: To isolate the major constituents and to investigate the antioxidant, antimicrobial, and cytotoxic activities of this herb. Materials and Methods: Methanolic extract of D. glaucum herb was fractionated using n-hexane, dichloromethane, and n-butanol. Butanol fraction was chromatographed using column chromatography and preparative thin layer chromatography to isolate the major constituents. Isolated compounds were elucidated by means of spectroscopic methods, including 1D, 2D NMR (1H, 13C, DEPT, COSY, HSQC, HMBC, NEOSY) and MS analysis. Total phenolic content using Folin–Ciocalteu reagent and antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay of the total methanolic extract were evaluated. Cytotoxic potential of both methanolic extract and butanol fraction was tested using a crystal violet viability assay. Antimicrobial activities of both extracts were investigated using diffusion agar technique. Results: Apigenin 6, 8-di-C-glucopyranoside (vicenin-2), quercetin-3'-O-methyl-3-O-glucopyranoside, quercetin-3'-O-methyl-3-O-galactopyranoside, quercetin-3-O-β-D-glucopyranoside, and quercetin-3-O-β-D-galactopyranoside were isolated and elucidated. Total phenolic content was (83.89 mg gallic acid equivalent/g extract). The EC50value of scavenging DPPH radical was 152.0 ± 2 μ g/mL. Butanol fraction showed the highest cytotoxic activity against cervical and breast carcinoma cells (IC50 3.6 and 6.1 μ g/mL, respectively). Both methanolic extract and butanol fraction showed wide spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria and some fungi. The highest activity was from methanolic extract against Enterococcus faecalis (83.25%) and against Candida tropicalis (77.03%) as compared to reference antibiotics. Conclusion: Data obtained from this study demonstrate that D. glaucum possesses significant antioxidant, cytotoxic, and antimicrobial activities which could be ascribed to its flavonoidal content.