Carbon dots have stimulated the curiosity of biomedical researchers due to their unique properties, such as less toxicity and high biocompatibility. The synthesis of carbon dots for biomedical application is a core area in research. In the current research, an eco-friendly hydrothermal technique was employed to synthesize high fluorescent, plant-derived carbon dots fromProsopis julifloraleaves extract (PJ-CDs). The synthesized PJ-CDs were investigated by physicochemical evaluation instruments such as fluorescence spectroscopy, SEM, HR-TEM, EDX, XRD, FTIR, and UV-Vis. The UV-Vis absorption peaks obtained at 270 nm due to carbonyl functional groups shifts of n→π*. In addition, a quantum yield of 7.88 % is achieved. The synthesized PJ-CDs showing the presence of carious functional groups O–H, C–H, C=O, O–H, C–N and the obtained particles in spherical shape with an average size of 8 nm. The fluorescence PJ-CDs showed stability against various environmental factors such as a broad range of ionic strength and pH gradient. The antimicrobial activity of PJ-CDs was tested against aStaphylococcus aureus, and aEscherichia coli. The results suggest that the PJ-CDs could substantially inhibit the growth ofStaphylococcus aureus. The findings also indicate that PJ-CDs are effective materials for bio-imaging inCaenorhabditis elegansand they can be also used for pharmaceutical applications.