Pistachio dehulling waste, known as Pistachio byproduct mixture (PBM), is a valuable resource that is often overlooked. An effective sustainable approach involves utilizing this agricultural waste through a fermentation process using edible filamentous fungi, demonstrating potential applications in nutrition and animal feed. The focus of this study was on converting PBM extract obtained from a hot water extraction pre-treatment into a protein-rich fungal biomass of Neurospora intermedia. The optimal conditions for growth were achieved at 72 h, pH 5.5, and 30 °C which are achieved by one-factor-at-a-time approach (OFAT), resulting in 6.7 g/L of dried fungal biomass, with a protein content of 20.4%. The conversion efficiency, expressed as grams of fungal biomass per gram of initial Total COD, was 0.37 g/g, highlighting the significant potential of PBM extract with high COD levels and low sugar content for fermentation processes. Additionally, an investigation was carried out to assess the impact of inoculation method, culture adaptation, COD/N ratio, and pH control on fungal biomass growth during cultivation. The results of optimal conditions with response of fungal biomass growth showed production of 0.44, 0.45, and 0.49 g of fungal biomass per gram of initial total COD, with protein contents of 20.2%, 27.1%, and 18.6%, respectively, leading to improved fungal biomass yield. The resulting protein-rich fungal biomass with a focus on the biorefinery platform to complete the value-added cycle, holds promise for applications in various sectors including food, animal feed, biochemical, and biomaterial industries.