A facile green hydrothermal method was developed for the preparation of fluorescent carbon dots (CDs) using apple juice as a raw material. The synthesized fluorescent CDs were characterized by UV–vis, fluorescence, Fourier transform infrared (FT-IR), dynamic light scattering (DLS), high resolution transmission electron microscopic (HR-TEM), life-time measurement and laser scanning confocal microscopic techniques. The CDs showed bright blue emission under UV-light (λ_ex=365nm). The CDs were used as alternative biocompatible fluorescent probes for imaging of bacteria (Mycobacterium tuberculosis and Pseudomonas aeruginosa) and fungal (Magnaportheoryzae). It was shown that the prepared CDs had no toxic effect on the both cells lines (bacteria and fungal), indicating that the CDs did not inhibit growth of bacterial and fungal cells, which confirms that the CDs exhibit good biocompatibility.