The current research focuses on the production and optimization of a natural yellowish-brown Azaphilone dye using Aspergillus niger. A variety of culture media were tested to ascertain the best conditions for dye synthesis. The formation of the yellowish-brown dye was confirmed by a color shift in the reaction mixture, and UV-Vis spectroscopy detected the dye at 450nm. Static conditions were found to be more favorable than shaking for higher dye yields, and fed-batch fermentation was more effective than batch fermentation. Maximum dye production was achieved after 28days of incubation. Factors such as temperature, pH, and inoculum percentage were shown to influence dye synthesis, with the highest production (2.5ml) occurring at 30°C, pH 7, and a 3% spore suspension in yeast extract peptone broth (YEPB) medium under static conditions. Gas chromatography-mass spectrometry (GC-MS) analysis validated the presence of Azaphilone dye in the culture filtrate. The dye was successfully applied to a pretreated cotton cloth. These findings advance our understanding of optimizing fungal dye production for sustainable and eco-friendly textile coloration applications. This study appears to be the first of its kind to report azaphilone dye production by A. niger in the YEPB medium.