Carbon nanodots (CNDs), reported as polyatomic carbon domains surrounded by amorphous carbon frames, have drawn extensive attention due to their easy-to-synthesis, outstanding electronic properties, and superior biocompatibility. However, substantial assessments regarding their biological performance are still needed, considering the complex nature of this type of relatively new nanoparticles. In this report, CNDs derived from urea and citric acid (U-CNDs) are investigated in the treatment of two cell lines, EA.hy926 and A549 cells, to examine the biocompatibility, cellular uptake, and antioxidation effect. The intracellular uptake study suggests an energy-dependent transport process into the cells mainly involving macropinocytosis and lipid raft-mediated endocytosis pathways. Moreover, the U-CNDs mostly target the mitochondria and present strong antioxidative effects by scavenging reactive oxygen species (ROS) in cells. Overall the findings in this report manifest that the U-CNDs could serve as a bioimaging reagent and antioxidant causing little deleteriousness in the respects of viability, plasma membrane integrity, and mitochondrial activity in both cell lines, and demonstrate some efficacy for inhibiting the metabolic activities of A549 cancer cells at higher concentration.