The combined biochemical impact of carbon, nitro-gen and temperature on the biosynthesis of the anti-fungal volatile organic compounds (VOCs): ethanol,2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol produced by Aureobasidium pullulans A1 and A3 was investigated using a Box–Behnken experimental design and response surface methodology (RSM). Normalized peak areas derived from solid phase micro extraction-gas chromatography–mass spectrometry (SPME-GC–MS) analysis, indicated that initial carbon content had a significant influence on the biosynthesis of ethanol and alcohols with greater than three carbon atoms. This result suggests a dominant activity of the A. pullulans anabolic pathway to biosynthesize three higher alcohols viade novo bio-synthesis of amino acids from sugar metabolism. Low concentrations of carbon (3–13 g l−1) with nitro-gen as both ammonium and amino acids in the growth medium resulted in a higher number of significant linear and quadratic relationships. Nitrogen availability and growth temperature had significant negative linear and quadratic correlations with VOCs biosynthesis in most instances. Isolate-dependant metabolic response was evident for all abiotic parameters tested on alcohol production. The findings of this study offer new perspectives to improve the production of key antifungal compounds by antagonists in biological control systems.