The aim of this study was to investigate the chemical composition and different biological activities of the small twigs (aerial parts before flowering stage) of Maclura spinosa (Willd.) C.C.Berg commonly called “Paper cup flower" family Moraceae. Phytochemical investigation of the of M. spinosa total methanolic extract (MSTME) by different chromatographic and spectroscopic techniques led to the isolation and identification of ten known compounds: arachidic acid methyl ester (1), lupeol acetate (2), glutinol (3), lupeol (4), β-sitosterol and stigmasterol (5), β-sitosterol-3-O-β-D-glucopyranoside (6), apigenin (7), 2,3-dihydrokaempferol (8), orobol (9) and Kaempferol-7-O-β-D-glucopyranoside (10). The gas chromatography-mass spectrometry (GC-MS) analysis of saponifiable and unsaponifiable matters led to the identification of eight and ten compounds, respectively. Moreover, total polyphenolic, total phenolic, and total flavonoid contents were determined by high-performance liquid chromatography (HPLC) analyses of MSTME. In the biological studies, the in-vitro antioxidant activity was determined by the DPPH method, n-but. (MSBF) and EtOAc fractions (MSEF) and MSTME showed the highest activity at 91.5%, 90.8%, and 88%, respectively at concentration 1 mg/mL with comparison to ascorbic acid and quercetin as positive controls. Also, the anti-inflammatory activity was assessed on RAW 264.7 cells, the MSTME (81.50%) and MSEF (48.46%) exhibited the highest activity at a concentration of 10 µg/mL in comparison with standard quercetin. On the other hand, the in-vivo antidiabetic activity was evaluated in streptozotocin (STZ) induced diabetic rats, at a daily oral dose of (300 mg/kg, p.o.) for 15 days, the MSTME and DCM fraction (MSDF) showed a significant reduction in blood glucose levels, total cholesterol and serum triglycerides (p < 0.01). The findings suggest the potential of M. spinosa as a natural product of the development of herbal medicine for the treatment of diabetes and inflammatory diseases.