This study conducted a comprehensive characterization and analyses on the fire-resistant behaviors of novel fungal fibers grown with substrate containing Silica (Si) source at multiple scales. At micro-scale, the results of SEM showed that silica affected the physiological activities of fungi, with the extent of effects depending upon its concentration. Fourier-transform infrared (FTIR) spectra displayed the existence of Si–O–C chemical bonds in fungal fibers grown with Si source, indicating that Si source becomes a part of the structure of fungal fibers. Thermogravimetric analysis (TGA) and Microscale combustion calorimetry (MCC) of fungal fibers exhibit an early thermal decomposition of non-combustible components, which will potentially help release the thermal stress and mitigation of spalling when used in concrete. Compared with polypropylene (PP) fibers, fungal fibers have a lower thermal degradation rate, a higher residual weight, a lower heat release peak temperature, and less total heat of combustion; all of these indicate improved thermal stability and fire resistance, and a lower rate of function loss in case of a fire. Additionally, the thermal stability and fire resistance of fungal fibers were improved with the increase of Si source concentration in the nutrition medium. For example, addition of 2% Si source in the feeding substrate leads to a 23.21% increase in residual weight in TGA, and a 23.66 W/g decrease in peak heat release rate as well as a 2.44 kJ/g reduction in total heat of combustion in MCC. At laboratory scale, compared with PP fibers, fungal fibers grown with 2% Si source have a higher residual weight of 40.40%, a higher ignition temperature of 200.50 °C, and a declined flame height of 11.64 mm in real fire scenarios. Furthermore, only in the fungal fibers grown with Si source, partial burning occurred. In post-fire conditions, the microstructure of residual char from fungal fibers grown with higher content of Si source became denser, which would lead to a reduction of the fuel vapor release and heat transfer. FTIR spectra of residual char demonstrated that fungal fibers grown with Si source formed more stable chemical bonds with higher heat of chemical bond formation, contributing to improved thermal stability and fire resistance. Therefore, compared with traditional fibers used for fiber reinforced concrete, incorporating the new natural grown fibers will potentially further improve the fire resistance of concrete and mitigate the concrete spalling.