Natural products are used as alternative medicines to treat various diseases including diabetes. Nauclea orientalis belongs to the family Rubiaceae and has been utilized as a traditional herbal for decades in many countries. The present investigation aimed to evaluate the antioxidant, α-amylase inhibitory, anti-hyperglycemic properties, and phytochemical contents in the ethanol (EtOH) Bark Extract of this plant. The EtOH Bark Extract showed that the IC50 value of DPPH scavenging activity was 20.69 ± 5.41 µg/mL whereas the standard ascorbic acid was 11.13 ± 2.81 µg/mL. The enzyme inhibitory potential of the extract against α-amylase was also determined with IC50 value of 242.2 ± 83.87 µg/mL. Compared to control animals, when administered at 400 mg/kg, the EtOH Bark Extract reduced blood glucose levels and body weight by 27.04% and 19.21%, respectively. N. orientalis bark extract reduced blood glucose and the area under the curve (AUC) after the oral glucose tolerance test. Phytochemical screening indicated the presence of carbohydrates, alkaloids, flavonoids, phenols, tannins, and saponins that might contribute to the biological activities from N. orientalis. In conclusion, this study demonstrates the EtOH Bark Extract of N. orientalis which possesses potent antioxidant, α-amylase inhibitory, and antihyperglycemic activity might be a potential candidate for biological and chemical analysis in pharmacy applications.