Trichoderma reesei and Aspergillus niger are efficient biological platforms for the production of various industrial products, including cellulases and organic acids. Nevertheless, despite the extensive research on these fungi, integrated analyses of omics-driven approaches are still missing. In this study, the intracellular metabolic profile of T. reesei RUT-C30 and A. niger N402 strains grown on glucose, lactose, carboxymethylcellulose (CMC), and steam-exploded sugarcane bagasse (SEB) as carbon sources for 48 h was analysed by proton nuclear magnetic resonance. The aim was to verify the changes in the primary metabolism triggered by these substrates and use transcriptomics data from the literature to better understand the dynamics of the observed alterations. Glucose and CMC induced higher fungal growth whereas fungi grown on lactose showed the lowest dry weight. Metabolic profile analysis revealed that mannitol, trehalose, glutamate, glutamine, and alanine were the most abundant metabolites in both fungi regardless of the carbon source. These metabolites are of particular interest for the mobilization of carbon and nitrogen, and stress tolerance inside the cell. Their concomitant presence indicates conserved mechanisms adopted by both fungi to assimilate carbon sources of different levels of recalcitrance. Moreover, the higher levels of galactose intermediates in T. reesei suggest its better adaptation in lactose, whereas glycolate and malate in CMC might indicate activation of the glyoxylate shunt. Glycerol and 4-aminobutyrate accumulated in A. niger grown on CMC and lactose, suggesting their relevant role in these carbon sources. In SEB, a lower quantity and diversity of metabolites were identified compared to the other carbon sources, and the metabolic changes and higher xylanase and pNPGase activities indicated a better utilization of bagasse by A. niger. Transcriptomic analysis supported the observed metabolic changes and pathways identified in this work. Taken together, we have advanced the knowledge about how fungal primary metabolism is affected by different carbon sources, and have drawn attention to metabolites still unexplored. These findings might ultimately be considered for developing more robust and efficient microbial factories.