Introduction:In recent years, the green synthesis of nanoparticles has received much attention. Green synthesis has several advantages over other methods: cost-effectiveness, simplicity, and non-toxicity. In the present study, we obtained the aqueous extract of Thymus daenensis (Celak) flora, biosynthesized the copper nanoparticles (Cu-NPs), and evaluated the antifungal activity. Methods: UV-vis spectroscopy analyses, scanning electron microscopy (SEM), and energy-dispersive X-ray (EDX) were used to identify the synthesized nanoparticles. The antifungal activity of the synthesized copper nanoparticles was evaluated using the microdilution method. Results: After adding the extract to the copper sulfate solution, the solution color changed from light blue to yellowish-green. A maximum peak at the wavelength of 414 nm confirmed the copper nanoparticles formation. Scanning electron microscopy demonstrated the particle size ranging from 30 nm to 42 nm. The biosynthesized Cu-NPs had an inhibitory effect against Candida albicans, Fusarium solani, Aspergillus Niger, and Aspergillus flavus. Conclusion: Our findings demonstrated that T. daenensis aqueous extract acts as a reducer and stabilizer factor. We successfully synthesized Cu-NPs from copper sulfate using T. daenensis (Celak) flora aqueous extract according to the UV-Vis spectrum, FTIR, and SEM results. This research was the first report of Cu-NPs synthesized from an aqueous T. daenensis (Celak) flora extract. Our simple, quick, and inexpensive method for biosynthesis of a nanoparticle, which showed antifungal activity, provides a new potential antifungal agent for therapeutic applications.