Background. Medicinal plants are widely used in many cultures, traditions, and civilizations worldwide. Plants with high contents of the valuable biological compounds can efficiently cure many diseases. This study is aimed at assessing, for the first time, the anti-α-amylase, antilipase, antimicrobial, and cytotoxic activities of Nonea obtusifolia (Willd.) DC. of five extracts from Palestine. Methods. The antimicrobial activity was estimated using well diffusion method for N. obtusifolia plant of five extracts against eight ATCC (American Type Culture Collection) and clinical isolates. The cytotoxic effects for these extracts were evaluated against HeLa (cervical) carcinoma cells using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Moreover, the lipase and α-amylase inhibitory properties were determined using standard biomedical assays. Results. The acetone extract of N. obtusifolia plant showed a more potent α-amylase inhibitory compared with acarbose with IC50 values of and , respectively. Additionally, the acetone and methanol extracts revealed moderate antilipase activity compared to orlistat with IC50 values of , , and , respectively. The methylene chloride extract was found to inhibit the growth of all the tested bacterial and fungal strains and also found to have potential cytotoxic effect against HeLa cancer cell line. Conclusion. This research work reports for the first time the biological activity of N. obtusifolia from Palestine, and the results were promising indicating that N. obtusifolia extracts contain valuable bioactive molecules that have a potential anti-α-amylase, antilipase, antibacterial, and antifungal cytotoxic potentials. Therefore, N. obtusifolia could have a medical significance in the future.