Cultured cell extracts from ten tropical strains of Aureobasidium pullulans were screened for antifungal activity against four pathogenic Aspergillus species (Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, and Aspergillus terreus) using the well diffusion and conidial germination inhibition assays. The crude cell extract from A. pullulans NRRL 58536 resulted in the greatest fungicidal activity against all four Aspergillus species and so was selected for further investigation into enhancing the production of antifungal activity through optimization of the culture medium, carbon source (sucrose and glucose) and amino acid (phenylalanine, proline, and leucine) supplementation. Sucrose did not support the production of any detectable antifungal activity, while glucose did with the greatest antifungal activity against all four Aspergillus species being produced in cells grown in medium containing 2.5 % (w/v) glucose. With respect to the amino acid supplements, variable trends between the different Aspergillus species and amino acid combinations were observed, with the greatest antifungal activities being obtained when grown with phenylalanine plus leucine supplementation for activity against A. flavus, proline plus leucine for A. terreus, and phenylalanine plus proline and leucine for A. niger and A. fumigatus. Thin layer chromatography, spectrophotometry, high-performance liquid chromatography, H-1-nuclear magnetic resonance, and MALDI-TOF mass spectrometry analyses were all consistent with the main component of the A. pullulans NRRL 58536 extracts being aureobasidins.