This study reports the synthesis and characterization of fungal carbon dots (F-CDs) derived from filamentous microfungi belonging to the subphylum Pezizomycotina. Carbon dots were synthesized from a cell-free aqueous fungal homogenate obtained from the mycelial biomass of Phialomyces macrosporus, Penicillium spp., and Fusarium sp. The water-soluble mycelial fraction, containing intracellular metabolites, soluble proteins, and other low-molecular-weight compounds released upon cell disruption, was used directly as a carbon precursor in a hydrothermal synthesis. The resulting F-CDs exhibited intense green fluorescence with excitation-dependent emission, as confirmed by UV–Vis absorption and photoluminescence spectroscopy. Transmission electron microscopy revealed spherical nanoparticles with an average diameter of 3.9 ± 1.1 nm and crystalline domains with an interplanar spacing of 0.26 nm. Zeta potential measurements indicated negatively charged surfaces (−15.6 to −18.4 mV), suggesting good colloidal stability and potential for biological interactions. Fluorescence microscopy demonstrated efficient uptake of F-CDs by Aspergillus niger hyphae, resulting in bright green staining and indicating high cellular compatibility. These results establish filamentous microfungi as previously unexplored and versatile carbon precursors for the sustainable production of green-emitting carbon dots with promising applications in bioimaging.