Fungi are promising sources of novel bioactive metabolites due to their abundant extracellular enzyme production, easy extraction, and purification. This study aimed to investigate the capability of some plant-associated fungi to produce L-asparaginase enzymes. Twenty plant-associated fungi were inoculated into a modified Czapex Dox broth medium containing 10.0 g/L L-asparagine and 0.3 mL of 2.5% phenol red (pH 7), and maintained at 25 ± 2 °C, for 7 days. The L-asparaginase enzyme activity was evaluated by measuring the amount of free ammonia released through the hydrolysis of L-asparagine through UV-visible spectrophotometry analysis at 450 nm. Seventy percent of the studied fungi could produce the L-asparaginase enzyme. Trichoderma atroviride, Aspergillus flavus, and T. harzianum demonstrated the highest production of L-asparaginase enzyme by 0.47, 0.35, and 0.24 U/mL, respectively. Furthermore, Cladosporium cladosporioides, C. ramotenellum and Verticillium dahliae with 0.022, 0.009 and 0.015 U/mL, respectively, showed the lowest production of the L-asparaginase enzyme among the others. This research also reports the first successful production of the L-asparaginase enzyme from Bipolaris oryzae, Curvularia trifolii, T. atroviride, T. harzianum, and T. virens. Plant-associated fungi represent a promising resource for the pharmaceutical industry due to their ability to produce bioactive compounds such as L-asparaginase. While their use may contribute to more sustainable and potentially cost-effective production, successful large-scale application depends on multiple factors beyond enzyme production, including cultivation feasibility, purification processes, and regulatory considerations.