Biochar-derived C-Dots from Picea, Molinia caerulea and Elaeis guineensis were synthesized through a hydrothermal process, and their physicochemical and optical characteristics and environmental effects were compared. These C-Dots were characterized by techniques such as Attenuated Total Reflection–Fourier Transform Infrared (ATR-FTIR), UV-Vis spectrophotometry, fluorescence spectroscopy, dynamic light scattering (DLS), Z potential, and High-Resolution Transmission Electronical Microscopy (HR-TEM). The ecotoxicity tests were performed using the Microtox™ test, making this study one of the few that use this method. The C-Dots from Molinia caerulea showed the best quantum yield (QY) of 8.39% and moderate ecotoxicity, while Elaeis guineensis has the lowest QY (2.31%) but with zero toxicity. Furthermore, the C-Dots from Picea presents good optical properties but showed high toxicity and limits its use. Finally, all C-Dots showed functional groups that could be biofunctionalized with biomolecules, especially C-Dots from Molinia caerulea and Elaeis guineensis show potential for use in the development of optical biosensors.