l-Asparaginase is a chemotherapeutic drug used in the treatment of acute lymphoblastic leukaemia (ALL), a malignant disorder in children. l-Asparaginase helps in removing acrylamide found in fried and baked foods that is carcinogenic in nature. l-Asparaginase is present in plants, animals and microbes. Various microorganisms such as bacteria, yeast and fungi are generally used for the production of l-asparaginase as it is difficult to obtain the same from plants and animals. l-Asparaginase from bacteria causes anaphylaxis and other abnormal sensitive reactions due to low specificity to asparagine. Toxicity and repression caused by bacterial l-asparaginase shifted focus to eukaryotic microorganisms such as fungi to improve the efficacy of l-asparaginase. Clinically available l-asparaginase has glutaminase and urease that may lead to side effects during treatment of ALL. Current work tested 45 fungal strains isolated from soil and agricultural residues. Isolated fungi were tested using conventional plate assay method with two indicator dyes, phenol red and bromothymol blue (BTB), and results were compared. l-Asparaginase activity was measured by cultivating in modified Czapek–Dox medium. Four strains have shown positive result for l-asparaginase production with no urease or glutaminase activity, among these C7 has high enzyme index of 1.57 and l-asparaginase activity of 33.59 U/mL. l-Asparaginase production by C7 was higher with glucose as carbon source and asparagine as nitrogen source. This is the first report focussing on fungi that can synthesize l-asparaginase of the desired specificity. Since the clinical toxicity of l-asparaginase is attributed to glutaminase and urease activity, available evidence indicates variants negative for glutaminase and urease would provide higher therapeutic index than variants positive for glutaminase and urease. © 2016, The Author(s).