Highly biocompatible, excellently photostable, nitrogen- and sulfur-containing novel zwitterionic carbon dots (CDs) were synthesized by microwave-assisted pyrolysis. The size of CDs were 2-5 nm, with an average size of 2.61 ± 0.7 nm. CDs were characterized by UV/vis spectroscopy, fluorescence spectroscopy, zeta potential, Fourier-transform infrared spectroscopy, X-ray diffraction, and time-resolved fluorescence spectroscopy. CDs were known to emit blue fluorescence when excited at 360 nm, that is, UV region, and emit in the blue region of visible spectrum, that is, at 443 nm. CDs showed excitation-independent photoluminescence behavior and were highly fluorescent even at lower concentration under UV light. These CDs were highly fluorescent in nature, with the quantum yield being as high as 80%, which is comparable to that of organic dyes. The CDs were further used to image two different oral cancer cell lines, namely, FaDu (human pharyngeal carcinoma) and Cal-27 (human tongue carcinoma). The cell viability assay demonstarted that CDs were highly biocompatible, which was further confirmed by the side scattering studies as no change in the granularity was observed even at the highest concentration of 1600 μg/mL. The generation of reactive oxygen species (ROS) was also investigated and negligible generaton of ROS was detected. In addition to that, the uptake phenomenon, cell cycle analysis, exocytosis, and cellular uptake at 4 °C and in the presence of ATP inhibitor were studied. It was found that CDs easily cross the plasma membrane without hampering the cellular integrity.