Carbon quantum dots (CQDs), a unique fluorescent carbon nanomaterial, have sparked widespread interest. They are highly biocompatible, water-soluble, chemically stable, and non-toxic. In this investigation, green-emissive CQDs were synthesized using orange juice as a precursor in a simple hydrothermal approach. The physicochemical investigation supports the particle size and functional groups of CQDs. The synthesized CQDs have emitted the bright green fluorescence light under the excitation wavelength of 420nm. They have exhibited good stability, high water solubility, and excitation-dependent characteristics. Moreover, the water soluble CQDs have excellent fluorescence quenching for Fe3+ ions, as well as outstanding selectivity and sensitivity. The biocompatibility test was carried out on MCF-7 cells, and the produced material demonstrated superior biocompatibility at 100μg/mL concentrations. The antibacterial activity was tested against four different bacteria. The S. aureus and K. pneumoniae bacteria have the best antibacterial activity compared to others. The antibacterial mechanism found that CQDs with abundant −NH3+ groups on their surface exhibit greater antibacterial activity. Also, it’s successfully prevents bacterial biofilms caused by E. coli, K. pneumoniae, S. aureus and B. subtilis. Moreover, the CQDs have been proven to be a nontoxic probe for multicolor imaging of MCF-7 cells. These findings indicate that the produced CQDs are effective multicolor fluorescent probes for MCF-7 cells and offer a new outlook for diagnostic tools in the biomedical field.