Carbon quantum dots (CQD) are an emergent nanomaterial with unique optical and biological properties. However, the purification of CQD is one of the bottlenecks that makes it difficult to scale for application in different areas. In this work, we explore for the first time the potential of centrifugal partition chromatography (CPC) as an alternative preparative technology to achieve the purification of CQD at the gram scale. The hydrothermal method was used to synthesize CQD from avocado peels. After 6 h at 250 degrees C, a complex mix of strong blue-fluorescent CQDs were obtained and submitted to CPC fractionation without pretreatment. The best results were obtained with the solvent system n-hexane-ethyl acetate-methanol-water (1:2:1:2, v/v/v/v), in an elution-extrusion protocol. Nine fractions were obtained and were characterized by UV-VIS spectrophotometry, Fourier transform infrared (F-TIR), and field emission scanning electron microscopy (FESEM), confirming the presence of CQD of different sizes. CPC fractionations indicate that a polarity-based separation mechanism can be used to purify CQD. Interestingly, four fractions showed antibacterial and anti-biofilm effects on Pseudomonas putida and Listeria monocytogenes. Therefore, CPC allows for better refining of this type of nanomaterial, and in combination with other techniques, it would serve to obtain CQD of higher purity, facilitating the physicochemical and bioactivity characterization of these particles. CPC would also allow the use of waste, such as avocado peels, to obtain new materials.