This study demonstrated an environmentally friendly, cost-effective, and easy method for manufacturing biocompatible fluorescent carbon quantum dots (CQDs) from the leaf extract of the medicinal herb Cissus quadrangularis. As-synthesized nitrogen doped-CQDs had fluorescence quantum yields of up to 5%. The presence of pyrrolic/pyridinic-N, quaternary-N (N–C3), and OC–N, as verified by X-ray photoelectron spectroscopy, reveals that nitrogen atoms were efficiently doped in CQDs. The size distribution of as-synthesized CQDs was found to be within 4–9 nm using a high-resolution transmission electron microscope. Meanwhile, nuclear magnetic resonance studies showed the presence of a significant proportion of sp2 and sp3 carbon groups. The zeta potential of as-synthesized CQDs was −7.8 mV, demonstrating the presence of a negatively charged surface with good stability in aqueous solution. Furthermore, the development of easy, affordable, and non-destructive fluorescence-based staining agents as an alternative to organic or synthetic dyes is widely sought. In this regard, as-synthesized N-doped CQDs shown remarkable fluorescent staining capabilities in this study and might be employed as a useful probe for optical and bio-imaging of bacteria, fungal, and plant cells. On the other hand, the cytotoxicity and cell viability of pine leaf, mushrooms, algae, fungus, and bacteria have also been examined. Even at higher doses (10.9 mg/ml), cell survival was demonstrated to be rather excellent and cytotoxicity was minimal for up to 4 days.