The present work aimed to investigate the phytochemical profile and biological activities of Sudanese baobab (Adansonia digitata L.) fruit pulp extract. Baobab fruit pulp serves as food, and has been used in traditional medicine in Africa for the treatment of several diseases, and believed to possess many biological activities. The sample of baobab fruit was collected from Blue Nile State in Sudan. The fruit pulp was obtained from baobab pods, sieved, extracted with methanol (80%), and analysed for its antioxidant activity, and alpha-glucosidase and nitric oxide (NO) inhibitory activities. The phytochemical constituents of the pulp were determined by LC-MS and 1H-NMR techniques. The results for antioxidant activity evaluated by DPPH and FRAP for methanolic extract were 232.70 mg Ascorbic Acid Equivalent Antioxidant Capacity (AEAC)/g and 222.28 mmol/g of Fe2+, respectively. Baobab pulp extract showed greater capability in inhibiting the generation of NO from the stimulated RAW264.7 cells at 98.45% inhibition and IC50 of 36.55 µg/mL; and α-glucosidase enzyme activity at 97.94% inhibition and IC50 of 58.59 µg/mL. The LC-MS/MS analysis of the baobab extract showed that 52 compounds were found in the baobab pulp, including 19 flavonoids, ten phenolic acids, four lipids (glycero-3-phosphocholine derivative, 1,2-didodecanoyl-sn-glycero-3-phospho-(1'-sn-glycerol), 1-pentadecanoyl-2-(13Z,16Z-docosadienoyl)-glycero-3-phosphate, and glycero-3-hosphocholine derivative), sugars, organic acid, iridoids, adenosine, scopoletin, and taraxerone. The NMR detected 21 metabolites from baobab pulp extract including carbohydrates, organic acids, vitamin, amino acids, phenolic compounds, alkaloids, and fatty acids. The present work is the first of its kind in comprehensively analysing the Sudanese baobab fruit pulp, combined with qualitative characterisation of chemical components using NMR, LC-MS, α-glucosidase, and NO inhibitors. Baobab fruit pulp contains several beneficial phytochemicals and biological activities, and it has potential positive effect on general human’s health.