The aim of this research is to improve the dyeability and antibacterial activity of silk fabric using natural fungal extract. The natural fungal pigment is extracted from the thermophilic fungi-basedspecies, namely Thermomyces, purified and characterized using Fourier transform infrared spectra, and subjected for the dyeing process. An experiment using Box and Behnken is designed with three levels and three variables using pH, temperature, and time as independent variables and with wash fastness, rubbing fastness, light fastness, and bacterial reduction (%) as dependent variables, and the conditions are optimized. Regression equations are obtained to analyze the fastness properties and bacterial reduction (%), and the optimum process parameters are identified. The result shows that the optimum conditions for dyeing the silk fabric by fungal pigmentation is 2% owm at 60°C for 30 min, maintaining a pH of 3.