Aims The aim of this study was to find new eukaryotic sources of the l-asparaginase (l-ASNase), since the prokaryotic sources of the enzyme are well-reported as causing allergic hypersensitivity reactions in a significant number of patients. This report describes screening for l-ASNase production by filamentous fungi isolated from the Brazilian Caatinga, and the optimization of fermentation parameters to increase fungal growth and improve yield in the production of l-ASNase. Methods and Results Thirty-two filamentous fungi were investigated in this study. When Aspergillus terreus strain S-18 was cultured in a proline-enriched medium, intracellular l-ASNase was expressed in concurrence with reduced l-glutaminase (l-GLUase) and protease activities. Fermentation conditions were then optimized in a 5-l bioreactor system to produce a maximum volumetric yield of 108 U total of l-ASNase activity. Conclusions The work reported here represents the first attempt to produce l-ASNase by filamentous fungi isolated from Brazil and offers a promising alternative eukaryotic source for l-ASNase production. Significance and Impact of the study In order to minimize the side effects caused by bacterial l-ASNase, the search of eukaryotic micro-organism for l-ASNase was carried out in fungi. This study demonstrates the diversity of filamentous fungi isolated from the Brazilian Caatinga Biome and the importance of knowledge of the microbial metabolism to obtain high concentrations of biotechnological products.