The edible filamentous fungus, Neurospora intermedia, has attracted significant attention over the past decade as a novel candidate for sustainable food and feed sources. This study aimed to evaluate the factors that enhance the content of B vitamins and carotenoid pigments in fungal biomass. We investigated the influence of physiological growth conditions, as well as other factors affecting the biosynthesis of these bioactive compounds, including precursors, metal cofactors, and stress conditions. Optimal conditions for biomass and carotenoid production were identified as 15 g/L glucose, 0.5 % (w/v) yeast extract, pH 6, and a 1 % (v/v) inoculum level in basic mineral medium. Among the metal cofactors tested, 5 mmol Mo2+ exhibited a highly significant effect on pigment production. The synthesis of B-group vitamins was influenced by disodium succinate after 72 h and 96 h, and by ribose after 72 h incubation. The maximum production levels recorded for biomass, carotenoids, and vitamins B1, B2, B3, and B6 were 4.90 g DM/L, 168.23 μg/g, 3.85, 12.49, 0.35, and 3.81 mg/g, respectively. This study highlights the biotechnological potential for producing biomass and bioactive compounds as viable resources for food and feed applications.