The toxicity of nanoparticles in a biological system is an integration of effects arising from surface functionality, particle size, ionic dissolution, etc. This complexity suggests that generalization of a material’s toxicity may be inappropriate. Moreover, from a medicinal point of view, toxicity can be used for treatment of malignant cells, such as cancer. In this study, highly biocompatible carbon nanodots (gCDs) were synthesized by reacting citric acid and urea in glycerol, which resulted in abundant hydroxyl functional groups on the particle surface. gCDs show excitation-dependent photoluminescence but with bright green to yellow emission. Importantly, a series of toxicity assessments showed that as-synthesized gCDs possessed exceptional biocompatibilities to various biological entities including 18 bacteria species, Petunia axillaris seedlings, and Artemia franciscana nauplii. Furthermore, the particles were shown to have low to no toxic effects on human embryonic kidney (HEK-293), breast (MCF-7), and oral squamous (CAL-27) carcinoma cell lines. Of particular interest, the gCDs displayed antiproliferative activities against ovarian choriocarcinoma cells (JAr/Jeg-3 cell lines), which may be further explored for cancer drug discovery.