This work pacts with a simple and environmentally friendly hydrothermal method to synthesize clove buds-derived carbon dots (CCDs) at different temperatures. Five CCDs samples were synthesized at different reaction temperatures ranging from 120 °C to 200 °C, respectively. Using UV–Visible (UV–Vis) and fluorescence spectroscopy (FL), the optical characteristics of CCDs made at various temperatures were examined and compared. The results show that the optical properties of prepared carbon dots (CDs) are significantly influenced by the temperature of carbonization. The cytocompatibility and hemolysis studies of optimized CCDs were performed to understand their interaction in the biological environment. The free radical scavenging capability of CDs was also explored. Further, in vitro cellular imaging of optimized CCDs200 was performed which shows strong and multicolour emission when excited with different laser sources. Interestingly, CDs are capable of brightening up RNA-rich nucleoli. Testing using ribonuclease digestion validates the localization of the CCDs in the nucleolus. Further, intracellular stability and good counterstaining compatibility feature them as a promising fluorescence probe for nucleolus imaging. Therefore, we have explored medicinal plant fruits as a carbon precursor for the synthesis of CDs without using any modifier which can be a potential candidate for multicolour nucleolus imaging and free radical scavenging applications.