Understanding how simple nutrient sources (carbon and nitrogen) shape fungal architecture is essential for generating tuneable mycelium-based materials. Here, we quantify the impact of defined nutrient environments on directive fungal growth in 2D and 3D systems. Using Aspergillus niger as a model, chemotrophic responses to simple sugars, sugar alcohols, and amino acids were measured using a miniaturised assay, generating chemotrophic index (CI) values under varying basal media compositions (low and high carbon and nitrogen). CI values were strongly influenced by nutrient type, enabling predictive insight into directional hyphal growth. Time-resolved fluorescence microscopy combined with AI-driven Imaris mapping was used to characterise 2D hyphal expansion under single and paired nutrient conditions (C–C, C–N, N–N). Results show that directional growth of A. niger can be guided towards simple biomolecules, with CI values providing good prediction across growth conditions. To extend these findings into 3D architectures, advanced imaging and AI-based mapping were applied to Botrytis cinerea, Rhizopus oryzae, and Trametes versicolor grown on individual sugars and defined sugar combinations. Distinct structural changes were observed in both single and combined carbon sources, including variations in branching frequency, filament depth, hyphal volume, and surface area. These observations highlight species-specific responses and reinforce the importance of controlled nutrient environments. Together, these results establish a quantitative framework linking nutrient inputs to fungal architecture across scales, enabling nutrient-driven design of mycelial materials with tailored properties for applications in textiles, biocomposites, and biotechnology.