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Herbal treatment for diabetes mellitus is widely used. The pharmacological activity is thought to be due to the phenolic compounds found in the plant leaves. The present study aims to investigate the phytochemical composition of <i>Urtica dioica</i> (UD) hydroethanolic extract and to screen its antidiabetic activity by disaccharidase hindering and glucose transport in Caco-2 cells. The results have shown that a total of 13 phenolic compounds in this work, viz. caffeic and coumaric acid esters (<b>1</b>, <b>2</b>, <b>4</b>–<b>7</b>, <b>10</b>), ferulic derivative (<b>3</b>), and flavonoid glycosides (<b>8</b>, <b>9</b>, <b>11</b>–<b>13</b>), were identified using HPLC-DAD-ESI/MS². The most abundant phenolic compounds were <b>8</b> (rutin) followed by <b>6</b> (caffeoylquinic acid III). Less predominant compounds were <b>4</b> (caffeoylquinic acid II) and <b>11</b> (kaempferol-O-rutinoside). The UD hydroethanolic extract showed 56%, 45%, and 28% (1.0 mg/mL) inhibition level for maltase, sucrase, and lactase, respectively. On the other hand, glucose transport was 1.48 times less at 1.0 mg/mL UD extract compared with the control containing no UD extract. The results confirmed that <i>U. dioica</i> is a potential antidiabetic herb having both anti-disaccharidase and glucose transport inhibitory properties, which explained the use of UD in traditional medicine.