Urtica dioica L. is one of the most widely utilized medicinal plants commonly applied in the form of tea, juice, and dietary supplements. This study aimed to assess the effect of the U. dioica ethanol–water extract (UdE) and polyphenolic fraction isolated from the extract (UdF) on normal human colon epithelial cells and to evaluate their protective activity against induced oxidative stress. The cytotoxic potential against human colorectal adenocarcinoma (HT29) and the anti-inflammatory effects were also investigated. UPLC-MS-DAD analysis revealed that both extracts were abundant in caffeic acid derivatives, specifically chlorogenic and caffeoylmalic acids, and therefore, they showed significant protective and ROS scavenging effects in normal human colon epithelial cells. Moreover, they had no negative impact on cell viability and morphology in normal cells and the extracts, particularly UdF, moderately suppressed adenocarcinoma cells. Furthermore, UdF significantly decreased IL-1β levels in HT29 cells. Our research indicates that U. dioica may provide significant health advantages because of its antioxidant and anti-inflammatory effects.