Carbon dots (CDs) are fluorescent nanoprobes offering a great potential in biological and medical applications due to their superior biocompatibility compared to metal chalcogenide quantum dots (e.g., CdSe). Key factors determining their cytotoxicity and cellular/intracellular tracking involve chemical nature and charge of surface functional groups. For the first time, we present a comprehensive cytotoxic study including cell cycle analysis of carbon dots differing in surface functionalization, namely pristine CDs (CDs-Pri) with negative charge due to carboxylic groups, polyethyleneglycol modified dots with neutral charge (CDs-PEG), and polyethylenimine coated dots with a positive charge (CDs-PEI). The CDs in vitro toxicity was studied on standard mouse fibroblasts (NIH/3T3). The results suggest that neutral CDs-PEG are the most promising for biological applications as they do not induce any abnormalities in cell morphology, intracellular trafficking, and cell cycle up to concentrations of 300 μg mL−1. Negatively charged CDs-Pri arrested the G2/M phase of the cell cycle, stimulated proliferation and led to higher oxidative stress, however they did not enter the cell nucleus. In contrast, positively charged CDs-PEI are the most cytotoxic, entering into the cell nucleus and inducing the largest changes in G0/G1 phase of cell cycle, even at concentrations of around 100 μg mL−1.